Entrapment of Radionuclides in Nanoparticle Compositions

ABSTRACT

The present invention is directed to the technical field of imaging compositions useful for diagnosing cancer and other diseases in a subject. In particular, the invention relates to a class of diagnostic compounds comprising a novel liposome composition with encapsulated metal entities such as radionuclides, for example  61 Cu and  64 Cu copper isotopes. The invention further relates to a novel method for loading delivery systems, such as liposome compositions, with metal entities such as radionuclides, and the use of liposomes for targeted diagnosis and treatment of a target site, such as cancerous tissue and, in general, pathological conditions associated with leaky blood vessels. The present invention provides a new diagnostic tool for the utilization of positron emission tomography (PET) imaging technique.

FIELD OF INVENTION

The present invention is directed to the technical field of imagingcompositions useful for diagnosing cancer and other diseases in asubject. In particular, the invention relates to a class of diagnosticcompounds comprising a novel liposome composition with encapsulatedradionuclides or metal entities, such as for example ⁶¹Cu and ⁶⁴Cucopper isotopes. The invention further relates to a novel method forloading delivery systems, such as liposome compositions, with metalentities such as radionuclides and the use of liposomes comprising metalentities such as radionuclides for targeted diagnosis and therapy of atarget site, such as cancerous tissue and, in general, pathologicalconditions associated with leaky blood vessels. The present inventionprovides a new diagnostic tool for the utilization of positron emissiontomography (PET) imaging technique.

BACKGROUND OF INVENTION

Liposomes can serve as vesicles to deliver a wide range of encapsulatedand/or membrane-incorporated therapeutic or diagnostic entities.Liposomes are usually characterized as nano-scale vesicles consisting ofan interior core separated from the outer environment by a membrane ofone or more bilayers. The bilayer membranes can be formed by amphiphilicmolecules e.g. synthetic or natural lipids that comprise a hydrophobicand a hydrophilic domain [Lasic, Trends Biotechnol., 16: 307-321, 1998].Bilayer membranes can also be formed by amphiphilic polymersconstituting particles (e.g. polymersomes and polymerparticles).

Liposomes can serve as carriers of an entity such as, withoutlimitation, a chemical compound, or a radionuclide, that is capable ofhaving a useful property or provide a useful activity. For this purpose,the liposomes are prepared to contain the desired entity in aliposome-incorporated form. The liposome incorporated entity can beassociated with the exterior surface of the liposome membrane, locatedin the interior core of the liposome or within the bilayer of theliposome. Methods for the incorporation of radionuclides into liposomesare e.g. surface labeling after liposome preparation [Phillips, Adv DrugDeliv Rev., 37: 13-32, 1999], label incorporation into the lipid bilayerof preformed liposomes [Morgan et al., J Med Microbiol., 14: 213-217,1981], surface labeling of preformed liposomes by incorporating lipidchelator conjugate during preparation [Goto et al., Chem harm Bull.(Tokyo), 37: 1351-1354, 1989; Seo et al., Bioconjucate Chem., 19:2577-2584, 2008], and aqueous phase loading of preformed liposome [Hwanget al., Biochim Biophys Acta., 716: 101-109, 1982; Phillips et al., IntJ Rad Appl Instrum B, 19: 539-547, 1992; Gabizon et al., J LiposomeRes., 1: 123-125, 1988; Henriksen et al., Nucl Med Bio., 31: 441-449,2004]. The incorporation of entities into liposomes by the aqueous phaseloading of preformed liposome is also referred to as “loading” andthereby “encapsulating” or “entrapping” the entities.

Encapsulating entities into the interior of liposomes through aqueousphase loading seems to provide the greatest in vivo stability, becauseof the protected location of the entity inside the liposome. The purposeof encapsulating an entity into a liposome is often to protect theentity from the destructive environment and rapid excretion in vivo. Theentrapment of the entity provides the opportunity for the encapsulatedentity to apply the activity of the entity mostly at the site or in theenvironment where such activity is advantageous but less so at othersites where the activity may be useless or undesirable. It is known thatliposomes having PEG chains attached to the outer surface have prolongedcirculation time in the blood stream. These liposome compositions caneffectively evade the immune system, which would otherwise attack theliposomes soon after injection causing fast clearance or rupture of theliposome and premature release of the agent entrapped inside. Byincreasing the blood circulation time, the agent entrapped in theliposome stays within the liposome until it reaches the target tissue.This phenomenon is referred to as passive targeting delivery, where anaccumulation of long-circulating nanoparticles in tumor areas orinflammatory sites is due to leaky vasculature and lack of an effectivelymphatic drainage system in these areas. For example, aradio-diagnostic entity entrapped within a long-circulating liposome canbe delivered by passive targeting to a diseased site within a subject tofacilitate a diagnosis thereof. Active- or ligand targeting deliverysystems is referred to liposome compositions with ligands attached onthe surface targeted against cell surface antigens or receptors [Allen,Science, 303: 1818-1822, 2004]. Combining the properties of targeted andlong-circulating liposomes in one preparation comprising a radionuclideencapsulated liposome composition would significantly enhance thespecificity and intensity of radioactivity localization in the targetsite e.g. a tumor. Ideally, such liposome compositions can be preparedto include the desired entity, e.g. a chemical compound or radionuclide,(i) with a high loading efficiency, i.e., high percentage ofencapsulated entity relative to the total amount of the entity used inthe encapsulation process, and (ii) in a stable form, i.e., with minimalrelease (i.e. leakage) of the encapsulated entity upon storage orgenerally before the liposome reaches the site or the environment wherethe liposome entrapped entity is expected to apply its intendedactivity.

Entrapment of radionuclides into nanoparticles such as liposomes can beobtained through use of chemical compounds called ionophores capable oftransporting metal ions across lipid membranes. Upon crossing themembrane barrier the radionuclide then binds preferably to a chelator,encapsulated in the interior of the liposome composition, due to itsstronger affinity thereto, allowing the release of free ionophore, andthe entrapment of the radionuclide in the liposome composition.

Copper isotopes are of great interest for use in diagnostic and/ortherapeutic application. For diagnostic applications this relates to thepositron-emitters ⁶¹Cu and ⁶⁴Cu, which can be used in positron emissiontomography (PET) diagnostic imaging. ⁶⁴Cu is an interesting copperisotope possessing all decay modalities, and with a half-life of 12.7 hit is favorable for biological studies. A half-life of about 6-12 happears to be ideal to allow for sufficient accumulation of liposome ininflammatory tissues or cancerous targets, yet providing enoughbackground clearance to permit early identification of the target[Gabizon et al., Cancer Res., 50: 6371-6378, 1990]. Furthermore, ⁶⁴Cucan be used as a model nuclide representing the chemical properties ofall copper isotopes.

Ideal radioisotopes for therapeutic applications are those with lowpenetrating radiation, such as β-, α- and auger electron-emitters.Examples of such radioisotopes are ⁶⁷Cu, ⁶⁷Ga, ²²⁵Ac, ⁹⁰Y, ¹⁷⁷Lu and¹¹⁹Sb. When the low energy emitting radioisotope in the form of aradiopharmaceutical reach the target site, the energy emitted is onlydeposited at the target site and nearby normal tissues are notirradiated. The energy of the emitted particles from the differentradioisotopes and their ranges in tissues will vary, as well as theirhalf-life, and the most appropriate radioisotope will be differentdepending on the application, the disease and the accessibility of thedisease tissue.

Ideal radioisotopes for diagnostic applications are those withrelatively short half-life, and those with high penetrating radiation tobe detected by imaging techniques such as positron emission tomography(PET) and/or single photon emission computed tomography (SPECT). Thehalf-life of the radionuclide must also be long enough to carry out thedesired chemistry to synthesize the radiopharmaceutical and long enoughto allow accumulation in the target tissue in the patient while allowingclearance through the non-target organs. The radionuclide, ⁶⁴Cu, hasproven to be a versatile isotope with respect to is applications in bothimaging [Dehdashti et al., J Nucl Med. 38: 103P, 1997] and therapy[Anderson et al., J Nucl Med., 36: 2315-2325, 1998].Radiopharmaceuticals and for example radiolabeled liposome compositionsconsisting of radionuclides, such as ⁶¹Cu (T %=3.33 h) and ⁶⁴Cu (T%=12.7 h) can be utilized for imaging by the positron emissiontomography (PET) technique, with the main advantages over single photonemission computed tomography (SPECT) being: a) employing annihilationcoincidence detection (ACD) technique whereby only photons detectedsimultaneously (<10⁻⁹ sec) by a pair of scinitillators opposite eachother are registered, instead of collimator, the sensitivity is markedlyimproved (×30-40) and the spatial resolution is enhanced by about afactor of two (<5 mm), since the detection field is (non-diverging)defined cylindrical volume and both the sensitivity and the resolutiondo not vary within the detection field [Kostarelos et al., J LiposomeRes., 9: 429-460, 1999]; b) PET scanners provide all images in the unitof radioactivity concentrations (e.g. Bq/ml) after corrections forphoton attenuation, scatters and randoms, thereby considering PET to bea more quantitative technique than SPECT [Seo, Curr. Radiopharm., 1:17-21, 2008].

The patent applications WO/2001/060417, WO/2004/082627, WO/2004/082626and US 20090081121, describe methods based on ionophoric loading ofradionuclides into liposomes. Further, the disclosed radionuclides whichare loaded into liposomes are heavy radionuclides and ¹¹C, ¹⁸F, ⁷⁶Br,⁷⁷Br, ⁸⁹Zr, ⁶⁷Ga, ¹¹¹In, ¹⁷⁷Lu, ⁹⁰Y and ²²⁵Ac. From a diagnosticstandpoint, these approaches are not useable for PET imagingapplications, but only SPECT, because of the limited use ofradionuclides.

Patent EP386 146 B1 describes a composition and method of use forliposome encapsulated compounds for neutron capture tumor therapy.However, these liposomes were loaded with stable elements (e.g. boron),that become radioactive only after activation.

In a theoretical study, Kostarelos et al., analyzed the therapeuticpotential of liposomes labeled with one of the radionuclides ¹³¹I, ⁶⁷Cu,¹⁸⁸Re or ²¹¹At, but chemical procedures for the preparation of thelabeled liposomes were not suggested [Kostarelos et al., J Liposome Res,9:407-424, 1999].

Only a few radiopharmaceuticals based on radioactive copper isotopes arediscovered and available today. Examples are ⁶⁰Cu-ATSM as hypoxiamarker, and ⁶⁴Cu-ATSM and ⁶⁴Cu-PTSM, which are suggested as potentialagents for tumor therapy. Further classes of substances arecopper-labeled peptides and antibodies in which the radioactive copperis linked to the biomolecule via a bifunctional chelator. There are noliposome compositions loaded with copper available for use asradiopharmaceuticals.

Several research groups have measured the permeability of anions andcations through lipid bilayers without the use of ionophores.

It is known in the field that the low ion permeability of phospholipidbilayers such as liposome compositions [Paula et al., Biophys. J.,74:319-327, 1998; Hauser et al., Nature, 239:342-344, 1972; Ceh et al.,J. Phys. Chem. B, 102:3036-3043, 1998; Mills et al., Biochim. Biophys.Acta, 1716:77-96, 2005; Papahadjopoulos et al., Biochim. Biophys. Acta,266:561-583, 1971; Puskin, J. Membrane Biol, 35:39-55, 1977] leads tohighly unfavorable loading kinetics for charged ion species. Thereforeit is common practice to use an ionophore to increase trans-bilayerdiffusion rates, and thereby improve or increase the loading ofmonovalent, divalent and trivalent cations into nanoparticles such asliposomes.

The patent application WO2006/043083 describes a method for loading ofradionuclides, which involves ionophores and chelators. It is mentionedin the application that a chelator may be an ionophore.

The patent application WO03/041682 discloses liposomes enclosingbiological agents. It is disclosed in the application thation-gradients, ionophores, pH gradients and metal complexationprocedures can be used for active loading of liposomes with biologicalagents. The application does not disclose a method for loading ofnanoparticles with metal entities wherein an osmotic gradient is used toincrease the loading efficiency or loading rate.

There is a need in the technical field of diagnostic applications toprovide various liposome compositions that are useful for delivery of avariety of compounds, for example radio-diagnostic and imaging entitiesuseful for PET.

SUMMARY OF INVENTION

The present invention relates to a novel and improved method forpreparation of metal entities and/or radionuclides encapsulated withinliposome compositions or nanoparticles. Contrary to what is commongeneral knowledge in the field, the inventors have found that loading ofmetal entities and/or radionuclides is efficient without the use ofionophores. Thus, in the new and inventive methods according to thepresent invention, the metal entities or radionuclides are loaded intothe nanoparticles without the use of an ionophore as a transportingmolecule.

Further, the presence of an osmotic stress on the membrane of thenanoparticles of the present invention has been found by the inventorsto improve the loading step of metal entities/radionuclides into theinterior of the nanoparticles. The positron-emitter ⁶⁴Cu is used as amodel nuclide representing the chemical properties of all copperisotopes.

The methods for preparation of a nanoparticle composition loaded withmetal entities wherein said methods do not involve the use of ionophoreaccording to the present invention comprise steps of:

a. Providing a nanoparticle composition comprising a vesicle formingcomponent and an agent-entrapping component enclosed by said vesicleforming component;

b. Entrapping (loading) the metal entities within the interior of thenanoparticle composition by enabling transfer of cation metal entitiesacross a membrane formed by the vesicle forming component by incubationof the nanoparticle composition in a solution comprising the metalentities.

Wherein said entrapping step involving incubation is understood as theloading of metal entities into the nanoparticle, such as the liposome.

According to the present invention, the loading efficiency or entrapmentof radionuclide is greater than 10%. Such a loading efficiency can be inthe range of 10% to 100%, preferably 80% to 100%, more preferably in therange of 95% to 100%.

According to one embodiment of the present invention, the incubationtemperature is lower than 100° C., such as for example in the range of10° C. to 80° C., such as 22° C. to 80° C., or such as 30° C. to 80° C.

The incubation time according to the present invention is a time periodshorter than 48 hours, such as between 1 min to 240 min, preferablybetween 1 min to 120 min and more preferably between 1 min to 60 min.

Metal entities according to the present invention may comprise orconsist of one or more radionuclides selected from the group consistingof Copper (⁶¹Cu, ⁶⁴Cu, and ⁶⁷Cu), Indium (¹¹¹In) Technetium (^(99m)Tc),Rhenium (¹⁸⁸Re), Gallium (⁶⁷Ga, ⁶⁸Ga), Lutetium (¹⁷⁷Lu), Actinium(²²⁵Ac) Yttrium (⁹⁰Y), Antimony (¹¹⁹Sb), Tin (¹¹⁷Sn, ¹¹³Sn), Dysprosium(¹⁵⁹Dy), Cobalt (⁵⁶Co), Iron (⁵⁹Fe), Ruthenium (⁹⁷Ru, ¹⁰³Ru), Palladium(¹⁰³Pd), Cadmium (¹¹⁵Cd) Tellurium (¹¹⁸Te, ¹²³Te), Barium (¹³¹Ba,¹⁴⁰Ba), Gadolinium (¹⁴⁹Gd, ¹⁵¹Gd), Terbium (¹⁶⁰Tb), Gold (¹⁹⁸Au, ¹⁹⁹Au),Lanthanum (¹⁴⁰La) and Radium (²²³Ra, ²²⁴Ra).

Metal entities according to the present invention may also comprise oneor more metal entities selected from the group of Gd, Dy, Ti, Cr, Mn,Fe, Co, Ni including divalent or trivalent ions thereof.

In one embodiment of the present invention, the method for preparationof nanoparticles involves a step wherein there is a difference inosmotic pressure between the exterior of the nanoparticles and theinterior of the nanoparticles during incubation, for example adifference of 5-800 mOsm/L, or preferably a difference of 5-100 mOsm/L.

The vesicle-forming component according to the present inventioncomprises one or more of the compounds selected from the groupconsisting of phospholipids, pegylated phospholipids and cholesterol,for example one or more amphiphatic compounds selected from the group ofHSPC, DSPC, DPPC, POPC, CHOL, DSPE-PEG-2000, DSPE-PEG-2000-RGD andDSPE-PEG-2000-TATE.

Agent-entrapping components according to the present invention areselected from the group consisting of chelators, reducing agents andagents that form low solubility salts with said radionuclides, forexample chelators selected from the group consisting of1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA),1,4,8,11-15 tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA),1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methanephosphonic acid)(DOTP), cyclam and cyclen.

The interior pH of the nanoparticles according to the present inventionis within the range of 4 to 8.5, such as 4.0 to 4.5, for example 4.5 to5.0, such as 5.0 to 5.5 for example 5.5 to 6.0, such as 6.0 to 6.5, forexample 6.5 to 7.0, such as 7.0 to 7.5, for example 7.5 to 8.0, such as8.0 to 8.5.

The stability of the radiolabeled nanoparticles provided by the presentinvention is such that less than 20% leakage of radioactivity isobserved for example less than 15% leakage, such as less than 12%leakage, for example less than 10% leakage, such as less than 8%leakage, for example less than 6% leakage, such as less than 4% leakage,for example less than 3% leakage, such as less than 2% leakage, forexample less than 1% leakage.

The present invention further provides kits of parts comprising:

a. A nanoparticle composition comprising i) a vesicle forming component,and ii) an agent-entrapping component enclosed by the vesicle formingcomponent; and

b. A composition containing a metal entity for loading into thenanoparticle,

Further, the present invention provides a nanoparticle compositionloaded with metal entities comprising:

i. a vesicle forming component,

ii. an agent-entrapping component enclosed by said vesicle-formingcomponent;

iii. a metal entity entrapped on the interior side of the nanoparticlecomposition.

In a particular embodiment of the present invention, the interior pH ofthe nanoparticle is within the range of 6 to 8.5, such as 6.0 to 6.5,for example 6.5 to 7.0, such as 7.0 to 7.5, for example 7.5 to 8.0, suchas 8.0 to 8.5.

The present invention further provides nanoparticle compositions for usein a method for treating, monitoring or diagnosis in a subject in need,such as for example in an imaging method which may be selected frompositron emission tomography (PET) scanning or single photon emissioncomputed tomography (SPECT) scanning and magnetic resonance imaging(MRI).

The present invention further provides nanoparticle compositionsprepared by the methods as disclosed by the invention.

DESCRIPTION OF DRAWINGS

FIG. 1: Separation of ⁶⁴Cu-Liposomes and free un-entrapped ⁶⁴Cu withsize exclusion chromatography (SEC) using a Sephadex G-25 column.Preformed liposomes consisting of DSPC/CHOL/DSPE-PEG₂₀₀₀ with DOTApre-encapsulated were loaded with ⁶⁴Cu using an incubation time of 60min and an incubation temperature of 50-55° C. achieving encapsulationefficiency as high as 96.7%.

FIG. 2: Separation of ¹⁷⁷Lu-Liposomes and free un-entrapped ¹⁷⁷Lu withsize exclusion chromatography (SEC) using a Sephadex G-25 column.Preformed liposomes consisting of DSPC/CHOL/DSPE-PEG₂₀₀₀ with DOTApre-encapsulated were loaded with ¹⁷⁷Lu using an incubation time of 60min and an incubation temperature of 50-55° C. achieving encapsulationefficiency of 81.0%.

FIG. 3: Loading efficiency of ⁶⁴Cu into liposomes as function ofincubation temperature without ionophore (dashed line) and withionophore (2HQ) (solid line). The loading efficiency of ⁶⁴Cu loaded intoliposomes without ionophore at 50-55° C. was 96.7%.

FIG. 4: Plot of standard curve and obtained results from a remoteloading experiment of Cu(II) into liposomes consisting of DSPC, CHOL andDSPE-PEG₂₀₀₀. The un-complexed Cu²⁺ was measured via an Cu(II)-selectiveelectrode and the achieved loading efficiency was

${{\left( {1 - \frac{1.2\mspace{14mu} {ppm}}{25\mspace{14mu} {ppm}}} \right) \cdot 100}\%} > {95{\%.}}$

Open squares denote Cu(II) standard curve in HEPES buffer, the crossdenotes HEPES 10 mM, 150 mM NaNO₃, pH 6.8, the open circle denotesunloaded liposomes and the closed circle denotes loaded liposomes.

FIG. 5: Structure of 1,2-Di-O-Hexadecyl-sn-Glycero-3-phosphocholine(1,2-Di-O-DPPC).

FIG. 6: Differential scanning calorimetry (DSC) scan ofDSPC/CHOL/DSPE-PEG₂₀₀₀ dispersion in HEPES buffer when mixturescontaining 10 mol % DSPE-PEG₂₀₀₀ and a) 20 mol % cholesterol and 70 mol% DSPC, b) 25 mol % cholesterol and 65 mol % DSPC c) 30 mol %cholesterol and 60 mol %, d) 35 mol % cholesterol and 55 mol % DSPC, e)40 mol % cholesterol and 50 mol % DSPC, f) 50 mol % cholesterol and 40mol % DSPC and g) Purified chelator-containing (10 mM DOTA) liposomesconsisting of DSPC/CHOL/DSPE-PEG₂₀₀₀ in the molar ratio 50:40:10.

FIG. 7: ⁶⁴Cu²⁺ loading efficiency into chelator containing liposomeswithout using ionophore as function of time at three differenttemperatures (50° C., 40° C. and 30° C.). The liposomes consist ofDSPC/CHOL/DSPE-PEG₂₀₀₀ in the molar ratio 50:40:10. The differencebetween the internal and external osmolarity of the liposomes was,Δ(mOsm/L)=+75 (higher internal osmolarity). The ratio between theinterior ⁶⁴Cu-DOTA complex and the un-encapsulated or non-complexed free⁶⁴Cu²⁺ is measured as ⁶⁴Cu-loading efficiency (%) using radio-thin layerchromatography (radio-TLC).

FIG. 8: ⁶⁴Cu²⁺ loading efficiency into chelator-containing liposomeswithout using ionophore as function of time at three differenttemperatures (50° C., 40° C. and 30° C.). The liposomes consist ofDSPC/CHOL/DSPE-PEG₂₀₀₀ in the molar ratio 50:40:10 and with equal intra-and extra-liposomal osmolarties. The ratio between the interior⁶⁴Cu-DOTA complex and the un-encapsulated or non-complexed free ⁶⁴Cu²⁺is measured as ⁶⁴Cu-loading efficiency (%) using radio-TLC.

DEFINITIONS

With the term “vesicle”, as used herein, we refer to an entity which ischaracterized by the presence of an internal void. Preferred vesiclesare formulated from lipids, including various amphiphatic componentsdescribed herein.

In various aspects the term “nanoparticles”, as used herein, areliposomes, polymersomes or other lipid or polymer shell structures thatconstitute a membrane in its broadest term surrounding a hydrous core.

With the term “chelator” and “chelating-agent” as used hereininterchangeably, we intend chemical moieties, agents, compounds, ormolecules characterized by the presence of polar groups able to form acomplex containing more than one coordinate bond with a transition metalor another entity. A chelator according to the present invention is awater soluble and/or non-lipophilic agent, and is thus not the same as a“lipophilic chelator” used for transportation of metal entities acrosslipophilic membranes such as vesicles formed by lipids.

With the term “metal entity” as used herein we intend a metal ion or aradionuclide, the latter used herein interchangeably with the termradioisotope.

With the term “phosphatide” we intend a phospholipid comprising aglycerol component.

With the term “amphiphatic” we intend a molecule which contains bothpolar and nonpolar regions.

With the term “binding affinity” and “affinity” as used hereininterchangeably, we refer to the level of attraction between molecularentities. Affinity can be expressed quantitatively as the dissociationconstant or its inverse, the association constant. In the context ofthis invention the affinity of a chelator or another agent-entrappingcomponent can relate to the binding affinity of the chelator DOTA for atransition metal ion or another metal entity, for example, Cu(II) orCu(I).

With the term “entrapped agent” we intend a metal isotope, which may bea radionuclide or a non-radioactive isotope, entrapped within a liposomecomposition or a nanoparticle composition as herein described.

With the term “agent-entrapping” as used herein, we refer to anycompound, without limitation, capable of trapping a metal ion or aradionuclide inside a liposome composition. Preferred agent-entrappingcomponents are chelating-agents, substances that have the ability toreduce other substances, referred to a reducing agent, or substancesthat form low solubility salts with radionuclides or metal entities.

With the terms “loading”, “encapsulation”, or “entrapment” as usedherein, are referred to an incorporation of radionuclides or metalentities into the interior of nanoparticle compositions. In the methodsof the present invention, this incorporation is done by incubation ofnanoparticle compositions with a solution comprising radionuclides ormetal entities.

With the terms “loading efficiency”, “entrapment efficiency” or“encapsulation efficiency” as used herein interchangeably, is referredto the fraction of incorporation of radionuclides or metal entities intothe interior of nanoparticle compositions expressed as a percentage ofthe total amount of radionuclide or metal entity used in thepreparation.

With the term “encapsulation stability”, “storage stability” or “serumstability” is referred to a stability test of the nanoparticlecomposition to measure the degree of leakage and/or release of theentrapped agent inside the nanoparticle composition.

With the term “radiolabeled complex” and the like, we refer to achelating agent and a radionuclide that form a complex.

With the term “targeting moiety” as used herein we intend saccharides,oligosaccharides, vitamins, peptides, proteins, antibodies andaffibodies and other receptor binding ligands characterized by beingattached to the nanoparticle surface through a lipid or polymercomponent for delivering the nanoparticles to a higher degree to thetarget site or into target cells.

The terms “drug”, “medicament”, “agent”, or “pharmaceutical compound” asused herein include, biologically, physiologically, or pharmacologicallyactive substances that act locally or systemically in the human oranimal body.

The terms “treating”, “treatment” and “therapy” as used herein referequally to curative therapy, prophylactic or preventative therapy andameliorating therapy. The term includes an approach for obtainingbeneficial or desired physiological results, which may be establishedclinically. For purposes of this invention, beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) condition, delay or slowing of progression or worsening ofcondition/symptoms, amelioration or palliation of the condition orsymptoms, and remission (whether partial or total), whether detectableor undetectable. The term “palliation”, and variations thereof, as usedherein, means that the extent and/or undesirable manifestations of aphysiological condition or symptom are lessened and/or time course ofthe progression is slowed or lengthened, as compared to notadministering compositions of the present invention.

The term “osmolarity” as used herein refers to the measure of soluteconcentration, defined as the number of osmoles (Osm) of solute perliter (L) of solution (Osm/L).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel and improved method forpreparation of metal entities and/or radionuclides encapsulated withinliposome compositions or nanoparticles which is based on an efficientloading temperature and favorable liposomal compositions. Further, thepresence of an osmotic stress of the membrane of the nanoparticles ofthe present invention has been found by the inventors to improve theloading step of metal entities/radionuclides into the interior of thenanoparticles.

The inventors have surprisingly found a method for loading chargedspecies (ions) into nanoparticles without adding ionophores to enhancetrans-membrane diffusion rates. Thus, the present invention discloses anovel method for fast entrapment of radionuclides (e.g. monovalent,divalent and trivalent cations) into liposome compositions withoutadding any lipophilic ionophores or any other carrier.

During the last 40 years lipophilic ionophores or complexes have beenused for enhancing the efficiency of encapsulating radionuclides (e.g.¹¹¹In³⁺, ¹⁷⁷Lu³⁺, ^(67/68)Ga²⁺, ^(99m)TcO₄ ⁻) in nanoparticles for invivo scintigraphic imaging and internal radiotherapeutic applications.The encapsulation (or loading) efficiencies using lipophilic ionophoreshave reached high levels as 90-95%. The present invention relates to anew method that does not use any lipophilic ionophores or other metalcarriers and can obtain similar or even higher loading efficiencies ofradionuclides into liposome compositions. A preparation method forloading metal entities and/or radionuclides into nanoparticles whichdoes not involve the use of ionophore has several advantages. Ionophoresmay be toxic to mammals, in particular to human beings. Therefore,nanoparticles prepared with the use of an ionophore will need to undergoextensive toxicity testing prior to regulatory approval. Furthermore,such nanoparticles will need to be purified prior to use to remove asmuch ionophore as possible and the extent of such purification will needto be monitored to ensure that the level of ionophore is below a certainthreshold.

Manufacture of nanoparticles of the present invention is done easilywith few components and without the need for extensive purification.When the nanoparticles of the present invention are administered topatients, the risks of side-effects such as toxicity or otherside-effects are reduced. Further, the novel preparation method allowfor an interior pH range of the nanoparticles which improve thestability of the nanoparticles. In this way, the use of thenanoparticles, methods or kits of part of the present invention isfacilitated since shelf-life, storage requirements and other aspectsrelated to the use of the present invention is improved compared to theprior art.

Also, while lipophilic ionophores are finding their usefulness forenhancing the efficiency of encapsulation of radionuclides e.g. cationsinto liposomes, the very lipophilic ionophores can also facilitate therelease of entrapped radionuclides from the liposomes. A release ofentrapped materials prematurely can result in not only an erroneousestimation of distribution of liposomes in vivo, but also a loss ofquality in the diagnostic images.

Further, the present invention solves a need in the technical field ofdiagnostic applications by providing nanoparticles for delivery of metalentities to tissues with pathological conditions associated with leakyblood vessels such as inflammatory sites or cancerous tissues.

Loading Efficiency and Loading Rate

The loading efficiency of loading methods for liposomes can be measuredby use of conventional methods in the art including ion-exchangechromatography, radio thin layer chromatography (radio-TLC), dialysis,or size exclusion chromatography (SEC) which can separate freeradioactive metal ions or free radiolabeled complexes from liposomeencapsulated radionuclides. When using SEC, the amount of radioactivityretained in liposomes compared to the amount of free radioactive metalions or free radiolabeled complexes can be determined by monitoring theelution profile during SEC and measuring the radioactivity with aradioactivity detector, or measuring the concentration of the metalentity using inductively coupled plasma mass spectrometry (ICP-MS),inductively coupled plasma atomic emission spectroscopy (ICP-AES) orinductively coupled plasma optical emission spectrometry (ICP-OES). Theradioactivity measured in the eluted fractions containing liposomescompared to eluted fractions not containing liposomes can be used todetermine the loading efficiency by calculating the percentage ofradioactivity retained in liposomes. Likewise, the amount ofradioactivity bound in liposomes can be compared to the amount ofradioactivity not entrapped in liposomes to obtain a measure of theloading efficiency when using other conventional methods known in theart.

The methods of the present invention ensure that a high amount of theradionuclides used in preparation will be entrapped within thenanoparticle. In one embodiment of the present method the efficiency ofloading is higher than 10%, such as in the range of 10%-100%, such ashigher than 15%, such as higher than 20%, such as higher than 25%, suchas higher than 30%, such as higher than 35%, for example higher than40%, such as higher than 50%, for example higher than 60%, such ashigher than 65%, for example higher than 70%, such as higher than 75%,for example higher than 80%, such as higher than 85%, for example higherthan 90%, such as higher than 95%, or such as higher than 96%, or suchas higher than 97%, or such as higher than 98%, or such as higher than99% or such as higher than 99.5% or such as higher than 99.9%. Inanother embodiment of the present invention the efficiency of loadingwhen using the methods of the present invention is higher than 30% whenassayed using size exclusion chromatography (SEC, described inexamples), ion-exchange chromatography or dialysis, such as 30% to 100%,including 55% to 100% loading efficiency, 80% to 100% loadingefficiency, and 95% to 100% loading efficiency.

Preferably, the efficiency of loading of the methods according to thepresent invention is in the range of 55% to 100% such as in the range of80% to 100%, more preferably in the range of 95% to 100%, such asbetween 95% to 97%, or such as between 97% to 99.9% loading efficiency.

The loading rate:

The loading of metals ions into liposomes can be divided into severalsteps including: (i) binding/coordination/adsorption of the ion to thelipid membrane, (ii) trans-membrane ion diffusion and (iii) binding ofions to the chelator. In the methods of the present invention, the lipidand chelator may be in large excess compared to the metal entities whichmay be for example, but not limited to, ⁶⁴Cu²⁺. In the example of ⁶⁴Cu²⁺the kinetics thus only depends on the ⁶⁴Cu²⁺ concentration. The rate ofcoordination/binding of Cu²⁺ to the membrane is rapid (likely to bediffusion limited) and binding of Cu²⁺ to the chelator (for exampleDOTA) occurs on timescale of seconds and can be verified by radio-TLC,or other conventional methods of the art. Since binding of metalentities to the membrane is fast, trans-membrane ion diffusion is themost probable rate limiting step.

In general, the rate of trans-membrane diffusion will depend on theconcentration gradient of the transported entity (according to Ficks1^(st) law), the membrane phase state (gel, fluid or liquid-ordered) andphysicochemical (hydrophilicity vs. hydrophobicity) properties of thetransported entity. These arguments substantiate the first orderequation (equation 1) presented below, which is here shown for ⁶⁴Cu²⁺,but is usable for other metal entities as well. The loading kinetics(example shown in FIG. 7-8) can be characterized by the equation

$\begin{matrix}{{\% \mspace{14mu} {load}} = {\frac{A_{{Cu} - {chelator}}}{A_{Cu} + A_{{Cu} - {chelator}} + A_{{Cu}{({ionophore})}}} = {a\left( {1 - {b\; ^{- {ct}}}} \right)}}} & \left( {{equation}\mspace{14mu} 1} \right)\end{matrix}$

where A_(Cu), A_(Cu-chelator) and A_(Cu(ionophore)) denote the TLCactivity of the ⁶⁴Cu²⁺, ⁶⁴Cu-Chelator and ⁶⁴Cu-ionophore specie. Thefitting parameter a, describes the plateau level (a˜100% if loadingproceeds according to 1^(st) order kinetics), b describes offset anduncertainty in t (b=1 when offset and uncertainties in t are small) andc describes the loading rate. By fitting of equation 1, each loadingprofile can be characterized by:(i) the initial velocity:

v _(ini) =a·b·c  (equation 2),

(ii) the time required to reach 95% loading:

t _((95%))=−ln((1−(95%)/a)/b)/c  (equation 3),

and (iii) the degree of loading reached at 60 min (% load_(1h)). Thelatter is directly comparable to the loading degree achieved using themethod based on SEC (see for example results in the examples andpresented in FIG. 3 and Tables 1, 2, 6 and 7).

The first order rate constant (c) depends on different parameters suchas temperature and osmolarity (see FIG. 7-8) (see next section) at whichthe loading is conducted. The initial velocity (V_(ini)), t_((95%)) and% load_(1h) are given in Table 8 for a set of loading conditions.

The loading rate of methods of the present invention can also bedescribed by the parameters initial velocity, the time required to reach95% loading and the degree of loading reached at 60 min.

Thus in one embodiment of the present invention, the initial velocity isin the range of 0.5%/min to 100%/min, preferably in the range of 3%/minto 100%/min and more preferably in the range of 23%/min to 100%/min.

In one embodiment of the present invention, the time required to reach95% loading is in the range of 0 minutes to 360 minutes, such as 1minutes to 240 minutes, preferably in the range of 5 minutes to 240minutes, such as in 5 minutes to 20 minutes, or such as in the range of9 minutes to 18 minutes.

In one embodiment of the present invention, the degree of loadingreached after 60 minutes is in the range of 10% to 100%, more preferablyin the range of 55% to 100%, such as the range of 80% to 100%, and evenmore preferably in the range of 95% to 100%, such as 95% to 99.9%.

Methods for loading of nanoparticles (such as liposomes) can be comparedby measuring parameters such as loading efficiency and loading ratesdescribed by the parameters initial velocity, the time required to reach95% loading and the degree of loading reached at 60 min. Thus, thesignificance of the contribution of ionophores to the above mentionedloading efficiency or loading rate can be determined by the methodsdisclosed herein.

The present invention provides a method for preparation of nanoparticles(such as liposomes) loaded with metal entities, wherein ionophores arenot used for loading of the nanoparticles, or wherein one or moreionophores are present in such small amounts that they do not contributesignificantly to the loading rate or the loading efficiency of theloading, since such methods essentially use the same mechanisms forloading as provided by the present invention. Thus, such methods caninclude methods wherein one or more ionophores are present in suchamounts that there is no significant increase in loading efficiencyand/or loading rate as determined by the parameters selected from thegroup of initial velocity, time required to reach 95% loading, degree ofloading reached at 60 min. Significance of differences in loading rateor loading efficiency can be calculated by using conventionalstatistical methods, such as for example Student t-test.

Nano-Particles

According to the embodiments of the invention, the liposome compositionis a micro-sized or a nano-sized particle that comprises a vesicleforming component and an agent-entrapping component. The vesicle formingcomponents form an enclosed barrier of the particle. Theagent-entrapping component may have at least one chemical moiety thatcontains one or more negatively charged groups or is capable of trappingions. The agent-entrapping component can furthermore be a reducingagent. The agent-entrapping component interacts with an encapsulatedagent, such as a metal entity comprising radio-diagnostic orradio-therapeutic agent, by electrostatic interaction, to form a stablecomplex or low soluble salt, or by reduction to form a precipitate. Thestabilization of the encapsulated agent, such as the radio-diagnostic orradio-therapeutic agent, prevents or minimizes the release of the agentfrom the vesicles in the blood circulation.

Agent entrapping components may further have at least one chemicalmoiety that contains one or more charged groups which may be negativelyor positively charged or is capable of trapping ions.

Metal Entities

Nanoparticles according to the present invention comprise metalentities. Metal entities according to the present invention may beselected from the metals known for a person skilled in the art andincluding any of the existing oxidation states for the metal, such asmonovalent cations, divalent cations, trivalent cations, tetravalentcations, pentavalent cations, hexavalent cations and heptavalentcations.

In one embodiment of the present invention, the metal entities arecations selected from the group of monovalent cations, divalent cations,trivalent cations, tetravalent cations, pentavalent cations, hexavalentcations and heptavalent cations, wherein divalent and trivalent cationsare preferred.

In one embodiment of the present invention, the metal entity is coppersuch as Cu(I) or Cu(II).

The nanoparticles of the present invention comprise entrapped metalentities, which may comprise or consist of metal radionuclides selectedfrom the group of isotopes consisting of Copper (⁶¹Cu, ⁶⁴Cu, and ⁶⁷Cu),Indium (¹¹¹In), Technetium (^(99m)Tc), Rhenium (¹⁸⁸Re), Gallium (⁶⁷Ga,⁶⁸Ga), Lutetium (¹⁷⁷Lu), Actinium (²²⁵Ac), Yttrium (⁹⁰Y), Antimony(¹¹⁹Sb), Tin (¹¹⁷Sn, ¹¹³Sn) Dysprosium (¹⁵⁹Dy), Cobalt (⁵⁶Co), Iron(⁵⁹Fe), Ruthenium (⁹⁷Ru, ¹⁰³Ru), Palladium (¹⁰³Pd), Cadmium (¹¹⁵Cd),Tellurium (¹¹⁸Te, ¹²³Te), Barium (¹³¹Ba, ¹⁴⁰Ba), Gadolinium (¹⁴⁹Gd,¹⁵¹Gd), Terbium (¹⁶⁰Tb), Gold (¹⁹⁸Au, ¹⁹⁹Au), Lanthanum (¹⁴⁰La), andRadium (²²³Ra, ²²⁴Ra), wherein said isotope of a metal radionuclide mayappear in any of the existing oxidation states for the metal. Theseoxidation states include monovalent cations, divalent cations, trivalentcations, tetravalent cations, pentavalent cations, hexavalent cationsand heptavalent cations.

In another embodiment, the entrapped metal entities comprise isotopesselected from the group of Rhenium (⁸⁶Re), Strontium (⁸⁹Sr), Samarium(¹⁵³Sm), Ytterbium (¹⁶⁹Yb), Thallium (²⁰¹Tl), Astatine (²¹¹At) whereinsaid isotope of a metal radionuclide may appear in any of the existingoxidation states for the metal. These oxidation states includemonovalent cations, divalent cations, trivalent cations, tetravalentcations, pentavalent cations, hexavalent cations and heptavalentcations.

In yet another embodiment, the entrapped metal entities compriseisotopes selected from the group of Copper (⁶¹Cu, ⁶⁴Cu, and ⁶⁷Cu),Indium (¹¹¹In), Technetium (^(99m)Tc), Rhenium (¹⁸⁸Re), Gallium (⁶⁷Ga,⁶⁸Ga), Actinium (²²⁵Ac), Yttrium (⁹⁰Y), Antimony (¹¹⁹Sb), and Lutetium(¹⁷⁷Lu), wherein said isotope of a metal radionuclide may appear in anyof the existing oxidation states for the metal. These oxidation statesinclude monovalent cations, divalent cations, trivalent cations,tetravalent cations, pentavalent cations, hexavalent cations andheptavalent cations.

In yet another embodiment of the present invention, one or more of theentrapped metal entities are selected from the group of metals which maybe used for magnetic resonance imaging (MRI) selected from the group ofconsisting of Gd, Dy, Ti, Cr, Mn, Fe, Fe, Co, Ni. Said metal entity mayappear in any of the existing oxidation states for the metal. Theseoxidation states include monovalent cations, divalent cations, trivalentcations, tetravalent cations, pentavalent cations, hexavalent cationsand heptavalent cations.

In a preferred embodiment of the present invention, one or more of theentrapped metal entities are selected from the group of consisting ofGd(III), Dy(III), Ti(II), Cr(III), Mn(II), Fe(II), Fe(III), Co(II),Ni(II).

Combinations of radionuclides are useful for simultaneousmonitoring/imaging and treatment of various diseases such as cancer,and/or for monitoring by use of several different imaging methods.Radionuclides and combinations of radionuclides may emit one or moretypes of radiation such as alpha particles, beta+ particles, beta−particles, auger electrons or gamma-rays. Combinations of radionuclidesmay further allow for one or more types of imaging and/or radiationtherapy. Thus, in another embodiment, this invention relates to vesiclesand methods for their preparation, wherein the vesicles comprise metalentities comprising two or more radionuclides, selected from the groupof Copper (⁶¹Cu, ⁶⁴Cu, and ⁶⁷Cu), Indium (¹¹¹In), Technetium (^(99m)Tc),Rhenium (¹⁸⁶Re, ¹⁸⁸Re), Gallium (⁶⁷Ga, ⁶⁸Ga), Strontium (⁸⁹Sr), Samarium(¹⁵³Sm), Ytterbium (¹⁶⁹Yb), Thallium (²⁰¹Tl), Astatine (²¹¹At), Lutetium(¹⁷⁷Lu), Actinium (²²⁵Ac), Yttrium (⁹⁰Y), Antimony (¹¹⁹Sb), Tin (¹¹⁷Sn,¹¹³Sn), Dysprosium (¹⁵⁹Dy), Cobalt (⁵⁶Co), Iron (⁵⁹Fe), Ruthenium (⁹⁷Ru,¹⁰³Ru), Palladium (¹⁰³Pd), Cadmium (¹¹⁵Cd), Tellurium (¹¹⁸Te, ¹²³Te),Barium (¹³¹Ba, ¹⁴⁰Ba), Gadolinium (¹⁴⁹Gd, ¹⁵¹Gd), Terbium (¹⁶⁰Tb), Gold(¹⁹⁸Au, ¹⁹⁹Au), Lanthanum (¹⁴⁰La), and Radium (²²³Ra, ²²⁴Ra), whereinsaid isotope of a metal radionuclide may appear in any of the existingoxidation states for the metal. These oxidation states includemonovalent cations, divalent cations, trivalent cations, tetravalentcations, pentavalent cations, hexavalent cations and heptavalentcations.

In a further embodiment, combinations of metal entities may include oneor more metals and one or more radionuclides which further allow for oneor more types of imaging and/or radiation therapy. Thus, in anotherembodiment, this invention relates to vesicles and methods for theirpreparation, wherein the vesicles comprise metal entities selected fromthe group of Gd, Dy, Ti, Cr, Mn, Fe, Fe, Co, Ni in any of the existingoxidation states for the metal, with radionuclides selected from thegroup of the group of Copper (⁶¹Cu, ⁶⁴Cu, and ⁶⁷Cu), Indium (¹¹¹In),Technetium (^(99m)Tc), Rhenium (¹⁸⁶Re, ¹⁸⁸Re), Gallium (⁶⁷Ga, ⁶⁸Ga),Strontium (⁸⁹Sr), Samarium (¹⁶³Sm), Ytterbium (¹⁶⁹Yb), Thallium (²⁰¹Tl),Astatine (²¹¹At), Lutetium (¹⁷⁷Lu), Actinium (²²⁵Ac), Yttrium (⁹⁰Y),Antimony (¹¹⁹Sb), Tin (¹¹⁷Sn, ¹¹³Sn), Dysprosium (¹⁵⁹Dy), Cobalt (⁵⁶Co),Iron (⁵⁹Fe), Ruthenium (⁹⁷Ru, ¹⁰³Ru), Palladium (¹⁰³Pd), Cadmium(¹¹⁵Cd), Tellurium (¹¹⁸Te, ¹²³Te), Barium (¹³¹Ba, ¹⁴⁰Ba), Gadolinium(¹⁴⁹Gd, ¹⁵¹Gd), Terbium (¹⁶⁰Tb), Gold (¹⁹⁸Au, ¹⁹⁹Au), Lanthanum (¹⁴⁰La),and Radium (²²³Ra, ²²⁴Ra), wherein said isotope of a metal radionuclidemay appear in any of the existing oxidation states for the metal. Theseoxidation states include monovalent cations, divalent cations, trivalentcations, tetravalent cations, pentavalent cations, hexavalent cationsand heptavalent cations.

Thus according to the present invention, nanoparticle compositions suchas vesicles may comprise one or more combinations selected from thegroup of ⁶⁴Cu and Gd(III), ⁶⁴Cu and Dy(III), ⁶⁴Cu and Ti(II), ⁶⁴Cu andCr(III), ⁶⁴Cu and Mn(II), ⁶⁴Cu and Fe(II), ⁶⁴Cu and Fe(III), ⁶⁴Cu andCo(II), ⁶⁴Cu and Ni(II), ⁶⁸Ga and Gd(III), ⁶⁸Ga and Dy(III), ⁶⁸Ga andTi(II), ⁶⁸Ga and Cr(III), ⁶⁸Ga and Mn(II), ⁶⁸Ga and Fe(II), ⁶⁸Ga andFe(III), ⁶⁸Ga and Co(II), ⁶⁸Ga and Ni(II), ¹¹¹In and Gd(III), ¹¹¹In andDy(III), ¹¹¹In and Ti(II), ¹¹¹In and Cr(III), ¹¹¹In and Mn(II), ¹¹¹Inand Fe(II), ¹¹¹In and Fe(III), ¹¹¹In and Co(II), ¹¹¹In and Ni(II),^(99m)Tc and Gd(III), ^(99m)Tc and Dy(III), ^(99m)Tc and Ti(II),^(99m)Tc and Cr(III), ^(99m)Tc and Mn(II), ^(99m)Tc and Fe(II), ^(99m)Tcand Fe(III), ^(99m)Tc and Co(II), ^(99m)Tc and Ni(II), ¹⁷⁷Lu andGd(III), ¹⁷⁷Lu and Dy(III), ¹⁷⁷Lu and Ti(II), ¹⁷⁷Lu and Cr(III), ¹⁷⁷Luand Mn(II), ¹⁷⁷Lu and Fe(II), ¹⁷⁷Lu and Fe(III), ¹⁷⁷Lu and Co(II), ¹⁷⁷Luand Ni(II), ⁶⁷Ga and Gd(III), ⁶⁷Ga and Dy(III), ⁶⁷Ga and Ti(II), ⁶⁷Gaand Cr(III), ⁶⁷Ga and Mn(II), ⁶⁷Ga and Fe(II), ⁶⁷Ga and Fe(III), ⁶⁷Gaand Co(II), ⁶⁷Ga and Ni(II), ²⁰¹Tl and Gd(III), ²⁰¹Tl and Dy(III), ²⁰¹Tland Ti(II), ²⁰¹Tl and Cr(III), ²⁰¹Tl and Mn(II), ²⁰¹Tl and Fe(II), ²⁰¹Tland Fe(III), ²⁰¹Tl and Co(II), ²⁰¹Tl and Ni(II), ⁹⁰Y and Gd(III), ⁹⁰Yand Dy(III), ⁹⁰Y and Ti(II), ⁹⁰Y and Cr(III), ⁹⁰Y and Mn(II), ⁹⁰Y andFe(II), ⁹⁰Y and Fe(III), ⁹⁰Y and Co(II) and ⁹⁰Y and Ni(II), wherein saidisotope of a metal radionuclide may appear in any of the existingoxidation states for the metal. These oxidation states includemonovalent cations, divalent cations, trivalent cations, tetravalentcations, pentavalent cations, hexavalent cations and heptavalentcations.

In a preferred embodiment, nanoparticle compositions such as vesiclesmay comprise one or more combinations of metal entities selected fromthe group consisting of ⁶⁴Cu and Gd(III), ⁶⁴Cu and Dy(III), ⁶⁴Cu andTi(II), ⁶⁴Cu and Cr(III), ⁶⁴Cu and Mn(II), ⁶⁴Cu and Fe(II), ⁶⁴Cu andFe(III), ⁶⁴Cu and Co(II), ⁶⁴Cu and Ni(II), ⁶⁸Ga and Gd(III), ⁶⁸Ga andDy(III), ⁶⁸Ga and Ti(II), ⁶⁸Ga and Cr(III), ⁶⁸Ga and Mn(II), ⁶⁸Ga andFe(II), ⁶⁸Ga and Fe(III), ⁶⁸Ga and Co(II), ⁶⁸Ga and Ni(II), ¹⁷⁷Lu andGd(III), ¹⁷⁷Lu and Dy(III), ¹⁷⁷Lu and Ti(II), ¹⁷⁷Lu and Cr(III), ¹⁷⁷Luand Mn(II), ¹⁷⁷Lu and Fe(II), ¹⁷⁷Lu and Fe(III), ¹⁷⁷Lu and Co(II), ¹⁷⁷Luand Ni(II) wherein said isotope of a metal radionuclide may appear inany of the existing oxidation states for the metal. These oxidationstates include monovalent cations, divalent cations, trivalent cations,tetravalent cations, pentavalent cations, hexavalent cations andheptavalent cations,

In an even more preferred embodiment, the nanoparticle compositions suchas vesicles may comprise one or more combinations of metal entitiesselected from the group consisting of ⁶⁴Cu and Gd(III), ⁶⁸Ga andGd(III), ¹⁷⁷Lu and Gd(III), ¹¹¹In and Gd(III), ⁶⁷Ga and Gd(III), ⁹⁰Y andGd(III), wherein the combinations of ⁶⁴Cu and Gd(III) and ⁶⁸Ga andGd(III) are most preferred.

Vesicles according to the present invention may comprise a combinationof one or more radionuclides for imaging and one or more radionuclidesfor therapy. Radionuclides for imaging comprise radionuclides such as⁶⁴Cu, ⁶¹Cu, ^(99m)Tc, ⁶⁸Ga, ⁸⁹Zr and ¹¹¹In.

Radionuclides for therapy comprise radionuclides such as ⁶⁴Cu, ⁶⁷Cu,¹¹¹In, ⁶⁷Ga, ¹⁸⁶Re, ¹⁸⁸Re, ⁸⁹Sr, ¹⁵³Sm, ¹⁶⁹Yb, ²⁰¹Tl, ²¹¹At, ¹⁷⁷Lu,²²⁵Ac, ⁹⁰Y, ¹¹⁹Sb, ¹¹⁷Sn, ¹¹³Sn, ¹⁵⁹Dy, ⁵⁶Co, ⁵⁹Fe, ⁹⁷Ru, ¹⁰³Ru, ¹⁰³Pd,¹¹⁵Cd, ¹¹⁸Te, ¹²³Te, ¹³¹Ba, ¹⁴⁰Ba, ¹⁴⁹Gd, ¹⁵¹Gd, ¹⁶⁰Tb, ¹⁹⁸Au, ¹⁹⁹Au,¹⁴⁰La, ²²³Ra and ²²⁴Ra.

In a preferred embodiment of the present invention, the vesicles ornanoparticles comprise two or more radionuclides selected from the groupof ⁶¹Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ²²⁵Ac, ⁹⁰Y, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re and¹¹⁹Sb.

An even more preferred embodiment of the present invention relates tovesicles or nanoparticles comprising ⁶⁴Cu and ¹⁷⁷Lu, or ⁶⁴Cu and ⁶⁷Cu,or ⁶¹Cu and ⁶⁷Cu, or ⁶⁴Cu and ⁹⁰Y, or ⁶⁴Cu and ¹¹⁹Sb, or ⁶⁴Cu and ²²⁵Ac,or ⁶⁴Cu and ¹⁸⁸Re, or ⁶⁴Cu and ¹⁸⁶Re, or ⁶⁴Cu and ²¹¹At, or ⁶⁴Cu and⁶⁷Ga, or ⁶¹Cu and ¹⁷⁷Lu, or ⁶¹Cu and ⁹⁰Y, or ⁶¹Cu and ¹¹⁹Sb, or ⁶¹Cu and²²⁵Ac, or ⁶¹Cu and ¹⁸⁸Re, or ⁶¹Cu and ¹⁸⁶Re, or ⁶¹Cu and ²¹¹At, or ⁶¹Cuand ⁶⁷Ga, or ⁶⁷Cu and ¹⁷⁷Lu, or ⁶⁷Cu and ⁹⁰Y, or ⁶⁷Cu and ¹¹⁹Sb, or ⁶⁷Cuand ²²⁵Ac, or ⁶⁷Cu and ¹⁸⁸Re, or ⁶⁷Cu and ¹⁸⁶Re, or ⁶⁷Cu and ²¹¹At, or⁶⁸Ga and ¹⁷⁷Lu, or ⁶⁸Ga and ⁹⁰Y, or ⁶⁸Ga and ¹¹⁹Sb, or ⁶⁸Ga and ²²⁵Ac,or ⁶⁸Ga and ¹⁸⁸Re, or ⁶⁸Ga and ¹⁸⁶Re, or ⁶⁸Ga and ²¹¹At, or ⁶⁸Ga and⁶⁷Cu.

Nanoparticles or vesicles comprising one or more radionuclides accordingto the present invention may be used for clinical imaging and/orradiotherapy. Clinical imaging includes imaging for diagnosis,monitoring the effects of treatment, or monitoring the location ofvesicles used for radiotherapy.

In a preferred embodiment, vesicles or nanoparticles of the presentinvention comprise a combination of radionuclides useful for combinedpositron emission tomography (PET) imaging and radiation therapy, suchas ⁶⁴Cu and ¹⁷⁷Lu, or such as ⁶⁴Cu and ⁶⁷Cu, or such as ⁶¹Cu and ⁶⁷Cu,or such as ⁶⁴Cu and ⁹⁰Y, or such as ⁶⁴Cu and ¹¹⁹Sb, or such as ⁶⁴Cu and²²⁵AC, or such as ⁶⁴Cu and ¹⁸⁸Re, or such as ⁶⁴Cu and ¹⁸⁶Re, or such as⁶⁴Cu and ²¹¹At.

In an even more preferred embodiment, vesicles or nanoparticles of thepresent invention comprise a combination of radionuclides useful forcombined positron emission tomography (PET) imaging and radiationtherapy, such as ⁶⁴Cu and ¹⁷⁷Lu.

According to the present invention, the nanoparticles may comprise oneor more isotopes different from copper which may be associated to theinner or outer surface of the nanoparticle composition via a linkermolecule such as a chelator. Such isotopes may be selected from thegroup of Indium (¹¹¹In) Technetium (^(99m)Tc), Rhenium (⁸⁶Re, ¹⁸⁸Re),Gallium (⁶⁷Ga, ⁶⁸Ga), Strontium (⁸⁹Sr), Samarium (¹⁵³Sm), Ytterbium(¹⁶⁹Yb), Thallium (²⁰¹Tl), Astatine (²¹¹At), Lutetium (¹⁷⁷Lu), Actinium(²²⁵Ac), Yttrium (⁹⁰Y), Antimony (¹¹⁹Sb), Tin (¹¹⁷Sn, ¹¹³Sn), Dysprosium(¹⁵⁹Dy), Cobalt (⁵⁶Co), Iron (⁵⁹Fe), Ruthenium (⁹⁷Ru, ¹⁰³Ru), Palladium(¹⁰³Pd), Cadmium (¹¹⁵Cd), Tellurium (¹¹⁸Te, ¹²³Te), Barium (¹³¹Ba,¹⁴⁰Ba), Gadolinium (¹⁴⁹Gd, ¹⁵¹Gd), Terbium (¹⁶⁰Tb), Gold (¹⁹⁸Au, ¹⁹⁹Au),Lanthanum (¹⁴⁰La), Radium (²²³Ra, ²²⁴Ra), Rhenium (¹⁸⁶Re), Strontium(⁸⁹Sr), Samarium (¹⁵³Sm), Ytterbium (¹⁶⁹Yb), Thallium (²⁰¹Tl) andAstatine (²¹¹At), wherein said isotope of a metal radionuclide mayappear in any of the existing oxidation states for the metal. Theseoxidation states include monovalent cations, divalent cations, trivalentcations, tetravalent cations, pentavalent cations, hexavalent cationsand heptavalent cations.

According to one embodiment of the present invention, the metal entitiescan be radionuclides selected from the group consisting of ⁶¹Cu, ⁶⁴Cu,⁶⁷Cu, ¹⁷⁷Lu, ⁶⁷Ga, ⁶⁸Ga, ²²⁵Ac, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹¹⁹Sb and ¹¹¹Inwherein said isotope of a metal radionuclide may appear in any of theexisting oxidation states for the metal. These oxidation states includemonovalent cations, divalent cations, trivalent cations, tetravalentcations, pentavalent cations, hexavalent cations and heptavalentcations.

In a preferred embodiment of the present invention, the metal entitiesare radionuclides selected from the group consisting of ⁶¹Cu, ⁶⁴Cu,⁶⁷Cu, ¹¹¹In and ¹⁷⁷Lu wherein said isotope of a metal radionuclide mayappear in any of the existing oxidation states for the metal. Theseoxidation states include monovalent cations, divalent cations, trivalentcations, tetravalent cations, pentavalent cations, hexavalent cationsand heptavalent cations.

In one embodiment of the present invention, the metal entities are twoor more radionuclides selected from the group consisting of ⁶⁴Cu and⁶⁷Cu, ⁶¹Cu and ⁶⁷Cu, ⁶⁴Cu and ⁹⁰Y, ⁶⁴Cu and ¹¹⁹Sb, ⁶⁴Cu and ²²⁵Ac, ⁶⁴Cuand ¹⁸⁸Re, ⁶⁴Cu and ¹⁸⁶Re, ⁶⁴Cu and ²¹¹At, ⁶⁴Cu and ⁶⁷Ga, ⁶¹Cu and¹⁷⁷Lu, ⁶¹Cu and ⁹⁰Y, ⁶¹Cu and ¹¹⁹Sb, ⁶¹Cu and ²²⁵Ac, ⁶¹Cu and ¹⁸⁸Re,⁶¹Cu and ¹⁸⁶Re, ⁶¹Cu and ²¹¹At, ⁶¹Cu and ⁶⁷Ga, ⁶⁷Cu and ¹⁷⁷Lu, ⁶⁷Cu and⁹⁰Y, ⁶⁷Cu and ¹¹⁹Sb, ⁶⁷Cu and ²²⁵Ac, ⁶⁷Cu and ¹⁸⁸Re, ⁶⁷Cu and ¹⁸⁶Re,⁶⁷Cu and ²¹¹At, ⁶⁸Ga and ¹⁷⁷Lu, ⁶⁸Ga and ⁹⁰Y, ⁶⁸Ga and ¹¹⁹Sb, ⁶⁸Ga and²²⁵Ac, ⁶⁸Ga and ¹⁸⁸Re, ⁶⁸Ga and ¹⁸⁶Re, ⁶⁸Ga and ²¹¹At, and ⁶⁸Ga and⁶⁷Cu.

In another embodiment of the present invention, the metal entities aretwo or more radionuclides selected from the group consisting of Copper(⁶¹Cu, ⁶⁴Cu, and ⁶⁷Cu), such as ⁶¹Cu and ⁶⁴Cu, or ⁶¹Cu and ⁶⁷Cu, or ⁶⁴Cuand ⁶⁷Cu, or ⁶¹Cu, ⁶⁴Cu and ⁶⁷Cu.

In one embodiment of the present invention, the metal entities areselected from the groups of metal entities as mentioned herein, whereinthe cations Hg²⁺ and Cu⁺, are excluded.

In a further embodiment of the invention, the radionuclide may also beentrapped within another carrier such as a nanoparticle that is usefulin diagnosing and/or treating a cancerous disease and, in general apathological condition associated with leaky blood vessels or anotherdisease in a subject.

A detailed description of exemplary vesicle forming components andagent-entrapping components for preparing the liposome compositions ofthe present invention are set forth below.

Vesicle Forming Component

A vesicle forming component is a synthetic or naturally-occurringamphiphatic compound which comprises a hydrophilic part and ahydrophobic part. Vesicle forming components include, for example, fattyacids, neutral fats, phosphatides, glycolipids, aliphatic alcohols, andsteroids. Additionally, vesicle forming components may further includelipids, diblock and triblock copolymers bolalipids, ceramides,sphingolipids, phospholipids, pegylated phospholipids and cholesterol.

In one embodiment of the present invention, the vesicle formingcomponents allow for a prolonged circulation time of the nanoparticles.

The vesicle forming component of the present invention or the method ofthe present invention may contain a hydrophilic polymer such as forexample a polyethylene glycol (PEG) component or a derivate thereof, ora polysaccharide. In such a case the vesicle forming component is saidto be derivatized with the hydrophilic polymer (e.g. PEG) or thepolysaccharide. In one embodiment, the polymer enables conjugation ofproteins or other receptor affinity molecules to the vesicle formingcomponent derivatized with the polymer. In another embodiment, theattachment of the polymer (e.g. PEG) to the liposome composition, allowsfor prolonged circulation time within the blood stream. Vesiclescomprising PEG chains on their surface are capable of extravasatingleaky blood vessels.

Examples of suitable vesicle forming lipids used in the presentinvention or the method of the present invention include, but are notlimited to: phosphatidylcholines such as1,2-dioleoyl-phosphatidylcholine, 1,2-dipalmitoyl-phosphatidylcholine,1,2-dimyristoyl-phosphatidylcholine, 1,2-distearoyl-phosphatidylcholine,1-oleoyl-2-palmitoyl-phosphatidylcholine,1-oleoyl-2-stearoyl-phosphatidylcholine,1-palmitoyl-2-oleoyl-phosphatidylcholine and1-stearoyl-2-oleoyl-phosphatidylcholine; phosphatidylethanolamines suchas 1,2-dioleoyl-phosphatidylethanolamine,1,2-dipalmitoyl-phosphatidylethanolamine,1,2-dimyristoyl-phosphatidylethanolamine,1,2-distearoyl-phosphatidylethanolamine,1-oleoyl-2-palmitoyl-phosphatidylethanolamine,1-oleoyl-2-stearoyl-phosphatidylethanolamine,1-palmitoyl-2-oleoyl-phosphatidylethanolamine,1-stearoyl-2-oleoyl-phosphatidylethanolamine andN-succinyl-dioleoyl-phosphatidylethanolamine; phosphatidylserines suchas 1,2-dioleoyl-phosphatidylserine, 1,2-dipalmitoyl-phosphatidylserine,1,2-dimyristoyl-phosphatidylserine, 1,2-distearoyl-phosphatidylserine,1-oleoyl-2-palmitoyl-phosphatidylserine,1-oleoyl-2-stearoyl-phosphatidylserine,1-palmitoyl-2-oleoyl-phosphatidylserine and1-stearoyl-2-oleoyl-phosphatidylserine; phosphatidylglycerols such as1,2-dioleoyl-phosphatidylglycerol, 1,2-dipalmitoyl-phosphatidylglycerol,1,2-dimyristoyl-phosphatidylglycerol,1,2-distearoyl-phosphatidylglycerol,1-oleoyl-2-palmitoyl-phosphatidylglycerol,1-oleoyl-2-stearoyl-phosphatidylglycerol,1-palmitoyl-2-oleoyl-phosphatidylglycerol and1-stearoyl-2-oleoyl-phosphatidylglycerol; pegylated lipids; pegylatedphospoholipids such asphophatidylethanolamine-N-[methoxy(polyethyleneglycol)-1000],phophatidylethanolamine-N-[methoxy(polyethyleneglycol)-2000],phophatidylethanolamine-N-[methoxy(polyethylene glycol)-3000],phophatidylethanolamine-N-[methoxy(polyethyleneglycol)-5000]; pegylatedceramides such asN-octanoyl-sphingosine-1-{succinyl[methoxy(polyethyleneglycol)1000]},N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]},N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethyleneglycol)3000]},N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethyleneglycol)5000]};lyso-phosphatidylcholines, lyso-phosphatidylethanolamines,lyso-phosphatidylglycerols, lyso-phosphatidylserines, ceramides;sphingolipids; glycolipids such as ganglioside GMI; glucolipids;sulphatides; phosphatidic acid, such as di-palmitoyl-glycerophosphatidicacid; palmitic fatty acids; stearic fatty acids; arachidonic fattyacids; lauric fatty acids; myristic fatty acids; lauroleic fatty acids;physeteric fatty acids; myristoleic fatty acids; palmitoleic fattyacids; petroselinic fatty acids; oleic fatty acids; isolauric fattyacids; isomyristic fatty acids; isostearic fatty acids; sterol andsterol derivatives such as cholesterol, cholesterol hemisuccinate,cholesterol sulphate, and cholesteryl-(4-trimethylammonio)-butanoate,ergosterol, lanosterol; polyoxyethylene fatty acids esters andpolyoxyethylene fatty acids alcohols; polyoxyethylene fatty acidsalcohol ethers; polyoxyethylated sorbitan fatty acid esters, glycerolpolyethylene glycol oxy-stearate; glycerol polyethylene glycolricinoleate; ethoxylated soybean sterols; ethoxylated castor oil;polyoxyethylene polyoxypropylene fatty acid polymers; polyoxyethylenefatty acid stearates; di-oleoyl-sn-glycerol;dipalmitoyl-succinylglycerol; 1,3-dipalmitoyl-2-succinylglycerol;1-alkyl-2-acyl-phosphatidylcholines such as1-hexadecyl-2-palmitoyl-phosphatidylcholine;1-alkyl-2-acyl-phosphatidylethanolamines such as1-hexadecyl-2-palmitoyl-phosphatidylethanolamine;1-alkyl-2-acyl-phosphatidylserines such as1-hexadecyl-2-palmitoyl-phosphatidylserine;1-alkyl-2-acyl-phosphatidylglycerols such as1-hexadecyl-2-palmitoyl-phosphatidylglycerol;1-alkyl-2-alkyl-phosphatidylcholines such as1-hexadecyl-2-hexadecyl-phosphatidylcholine;1-alkyl-2-alkyl-phosphatidylethanolamines such as1-hexadecyl-2-hexadecyl-phosphatidylethanolamine;1-alkyl-2-alkyl-phosphatidylserines such as1-hexadecyl-2-hexadecyl-phosphatidylserine;1-alkyl-2-alkyl-phosphatidylglycerols such as1-hexadecyl-2-hexadecyl-phosphatidylglycerol;N-Succinyl-dioctadecylamine; palmitoylhomocysteine;lauryltrimethylammonium bromide; cetyltrimethyl-ammonium bromide;myristyltrimethylammonium bromide; N-[1,2,3-dioleoyloxy)-propyl]-N,N,Ntrimethylammoniumchloride(DOTMA); 1,2-dioleoyloxy-3(trimethyl-ammonium)propane(DOTAP); and1,2-dioleoyl-c-(4′-trimethylammonium)-butanoyl-sn-glycerol (DOTB).

Such examples of suitable vesicle forming lipids used in the presentinvention or the methods of the present invention further includehydrogenated soy phosphatidylcholine (HSPC).

In one embodiment the vesicle forming component include compoundsselected from the group of DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine), CHOL (Cholesterol),DSPE-PEG-2000(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), POPC(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DSPE-PEG₂₀₀₀-TATE,(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]-TATE).

In one preferred embodiment the vesicle forming component includecompounds selected from the group of DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine), CHOL (Cholesterol),DSPE-PEG-2000(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine),DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DSPE-PEG₂₀₀₀-TATE,(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]-TATE) and hydrogenated soy phosphatidylcholine (HSPC).

In one embodiment of the nanoparticle composition, the vesicle formingcomponent consists of amphiphatic compounds selected from the groupconsisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) “A”,cholesterol “B”, and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG-2000) “C” in the molar ratio of A:B:C, wherein Ais selected from the interval 45 to 65, B is selected from the interval35 to 45, and C is selected from the interval 2 to 20 and whereinA+B+C=100.

In one preferred embodiment of the nanoparticle composition, the vesicleforming component consists of amphiphatic compounds selected from thegroup consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)“A”, cholesterol “B”, and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG-2000) “C” in the molar ratio of A:B:C, wherein Ais selected from the interval 45 to 65, B is selected from the interval35 to 45, and C is selected from the interval 1 to 20 and whereinA+B+C=100.

In another preferred embodiment the vesicle forming component includeDSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), CHOL (Cholesterol),DSPE-PEG-2000(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]) in a molar ratio of 50:40:10.

In another embodiment of the disclosed method, the vesicle formingcomponent consists of amphiphatic compounds selected from the groupconsisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) “A”,cholesterol “B”, and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG-2000) “C”, and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]-TATE (DSPE-PEG-2000-TATE) “D” with the molar ratioA:B:C:D, wherein A is selected from the interval 45 to 65, B is selectedfrom the interval 35 to 45, C is selected from the interval 5 to 13, Dis selected from the interval 0 to 3, and wherein A+B+C+D=100.

The radiolabeled nanoparticle composition mentioned above may furthercomprise a targeting moiety enabling the nanoparticle to specificallybind to target cells bearing the target molecule, or a moietyspecifically binding to diseased target. The targeting moiety may beattached to the surface of the nanoparticle composition via alipid-anchoring molecule or a PEG-conjugated lipid component.

The vesicle forming component may further comprise a lipid-conjugate ofan antibody or an affibody or a peptide that acts as a targeting moietyto enable the nanoparticle to specifically bind to target cell bearing atarget molecule.

The vesicle forming component may also consist of a lipid-conjugate ofan antibody or an affibody or a peptide that acts as a targeting moietyto enable the nanoparticle to specifically bind to diseased target.

The antibodies useful in the present invention may be monospecific,bispecific, trispecific, or of greater multi-specificity. For example,multi-specific antibodies may be specific for different epitopes of acytokine, cell, or enzyme which may be present in an increased amount atthe target site compared to the normal tissues.

An “antibody” in accordance with the present specification is defined asa protein that binds specifically to an epitope. The antibody may bepolyclonal or monoclonal. Examples of monoclonal antibodies useful inthe present invention is selected from the group consisting of, but notlimited to, Rituximab, Trastuzumab, Cetuximab, LymphoCide, Vitaxin,Lym-1 and Bevacizumab. In a preferred embodiment, the monoclonalantibodies are selected from the group consisting of Rituximab,Trastuzumab, Cetuximab, LymphoCide, Vitaxin, Lym-1, and Bevacizumab.

An “affibody” is defined as a small and stable antigen-binding moleculethat can be engineered to bind specifically to a large number of targetproteins. The affibody molecules mimic monoclonal antibodies in manyways, and in addition offer several unique properties making them asuperior choice for a number of applications. These applications includeincorporating the affibodies as lipid-conjugates in liposomecompositions targeted for a tissue or a cell in a neovascular orinflammatory site, wherein the radionuclide, such as a copper isotope,but not limited to, ⁶¹Cu, ⁶⁴Cu and ⁶⁷Cu, is included for diagnosticand/or therapeutic applications. Examples of affibody molecules usefulin the present invention is collected for the group consisting of, butnot limited to, anti-ErbB2 affibody molecule and anti-Fibrinogenaffibody molecule.

The peptides useful in the present invention act as a targeting moietyto enable the nanoparticle to specifically bind to a diseased target,wherein the peptides are selected from the group consisting of, but notlimited to, RGD, somatostatin and analogs thereof, and cell-penetratingpeptides. In one embodiment, the peptides are selected from the groupconsisting of RGD, somatostatin and analogs thereof, andcell-penetrating peptides. In one embodiment, the somatostatin analog isoctreotate (TATE).

The vesicle forming components are selected to achieve a specifieddegree of fluidity or rigidity, to control the stability of the liposomecompositions in vivo and to control the rate of release of the entrappedagent inside the liposome composition. The rigidity of the liposomecomposition, as determined by the vesicle forming components, may alsoplay a role in the fusion or endocytosis of the liposome to a targetedcell.

The surface charge of the vesicles may also be an important factor inthe loading of the vesicle, for controlling the stability of theliposome compositions in vivo and to control the rate of release of theentrapped agent inside the liposome composition. Thus according to thepresent invention, the vesicle forming components may further beselected in order to control the surface charge of the formed vesicles.

Agent-Entrapping Component

The agent-entrapping component of the present invention or the method ofthe present invention may be a chelating agent that forms a chelatingcomplex with the transition metal or the radiolabeled agent, such as theradionuclide.

When a chelator (such as for example DOTA) is present in the aqueousphase of the liposome interior, the equilibrium between the exterior andthe interior of the liposome is shifted since metal ions that pass themembrane barrier are effectively removed from the inner membrane leafletdue to tight binding to the chelator. The very effective complexformation of the metal ion with the chelator renders the free metal ionconcentration in the liposome interior negligible and loading proceedsuntil all metal ions have been loaded into the liposome or equilibriumhas been reached. If excess of chelator is used, the metal ionconcentration in the liposomes will be low at all stages during loadingand the trans-membrane gradient will be defined by the free metal ionconcentration on the exterior of the liposomes.

According to the present invention, chelators may be selected from thegroup comprising 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (DOTA) and derivatives thereof; 1,4,8,11-tetraazacyclotetradecane(cyclam) and derivatives thereof; 1,4,7,10-tetraazacyclododecane(cyclen) and derivatives thereof;1,4-ethano-1,4,8,11-tetraazacyclotetradecane (et-cyclam) and derivativesthereof; 1,4,7,11-tetraazacyclotetradecane (isocyclam) and derivativesthereof; 1,4,7,10-tetraazacyclotridecane ([13]aneN₄) and derivativesthereof; 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (DO2A) andderivatives thereof; 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid(DO3A) and derivatives thereof;1,4,7,10-tetraazacyclododecane-1,7-di(methanephosphonic acid) (DO2P) andderivatives thereof;1,4,7,10-tetraazacyclododecane-1,4,7-tri(methanephosphonic acid) (DO3P)and derivatives thereof;1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methanephosphonic acid)(DOTP) and derivatives thereof; ethylenediaminetetraacetic acid (EDTA)and derivatives thereof; diethylenetriaminepentaacetic acid (DTPA) andderivatives thereof;1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) andderivatives thereof, or other adamanzanes and derivates thereof.

In another embodiment, the agent-entrapping component according to thepresent invention may be a substance that has the ability to reduceother substances, thus referred to as a reducing agent. Examples ofreducing agents comprise ascorbic acid, glucose, fructose,glyceraldehyde, lactose, arabinose, maltose and acetol.

In one embodiment of the present invention the loaded copper isotope,which may be Cu(II) or Cu(I) cations, is reduced to a lower oxidationstate upon diffusion through the vesicle membrane, thus trapping thecopper isotope within the vesicle. In another embodiment, theradionuclide different from copper, is reduced to a lower oxidationstate upon diffusion through the vesicle membrane, thus trapping theradionuclide different from copper within the vesicle.

In a further embodiment, an agent-entrapping component within the scopeof the present invention or the method of present invention may be asubstance with which the radionuclide or metal entity, such as copperisotope, forms a low solubility salt. Examples of such are copperphosphates, copper oxalate and copper chlorides. In one embodiment, thelow solubility salt formed with copper (Cu(II) or Cu(I)) is selectedfrom the group consisting of copper phosphates, copper oxalate andcopper chlorides.

In one embodiment of the present invention or the method of the presentinvention the agent-entrapping component is a chelator selected from thegroup consisting of macrocyclic compounds comprising adamanzanes;1,4,7,10-tetraazacyclododecane ([12]aneN₄) or a derivative thereof;1,4,7,10-tetraazacyclotridecane ([13]aneN₄) or a derivative thereof;1,4,8,11-tetraazacyclotetradecane ([14]aneN₄) or a derivative thereof;1,4,8,12-tetraazacyclopentadecane ([15]aneN₄) or a derivative thereof;1,5,9,13-tetraazacyclohexadecane ([16]aneN₄) or a derivative thereof;and other chelators capable of binding metal ions such asethylene-diamine-tetraacetic-acid (EDTA) or a derivative thereof,diethylene-triamine-penta-acetic acid (DTPA) or a derivative thereof.

In one embodiment of the present invention or the method of the presentinvention the agent-entrapping component is a chelator selected from thegroup consisting of 1,4-ethano-1,4,8,11-tetraazacyclotetradecane(et-cyclam) or a derivative thereof; 1,4,7,11-tetraazacyclotetradecane(iso-cyclam) or a derivatives thereof;1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or aderivative thereof; 2-(1,4,7,10-tetraazacyclododecan-1-yl)acetate (DO1A)or a derivative thereof; 2,2′-(1,4,7,10-tetraazacyclododecane-1,7-diyl)diacetic acid (DO2A) or a derivative thereof;2,2′,2″-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid(DO3A) or a derivative thereof;1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methanephosphonic acid)(DOTP) or a derivative thereof;1,4,7,10-tetraazacyclododecane-1,7-di(methanephosphonic acid) (DO2P) ora derivative thereof;1,4,7,10-tetraazacyclododecane-1,4,7-tri(methanephosphonic acid) (DO3P)or a derivative thereof; 1,4,8,11-15tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) or aderivative thereof; 2-(1,4,8,11-tetraazacyclotetradecane-1-yl)aceticacid (TE1A) or a derivative thereof;2,2′-(1,4,8,11-tetraazacyclotetradecane-1,8-diyl)diacetic acid (TE2A) ora derivative thereof; and other adamanzanes or derivates thereof.

In one embodiment of the present invention or the method of the presentinvention the agent-entrapping component is selected from the groupconsisting of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(DOTA) or a derivative thereof, 1,4,8,11-15tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) or aderivative thereof,1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methanephosphonic acid)(DOTP), cyclam and cyclen.

In a particularly important embodiment of the present invention ormethod of the present invention, the agent-entrapping component is1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

Ionophores

Ionophores can be characterized as ion-transporters, lipophilicchelators, channel formers, lipophilic complexes etc. In general anionophore can be defined as a lipid-soluble molecule that transportsions across the lipid bilayer of cell membranes or liposomes. Ionophoresare used to increase permeability of lipid membranes to ions andfacilitate transfer of molecules through, into and out of the membrane.There are general two broad classifications of ionophores, where one is;chemical compounds, mobile carriers or lipophilic chelators that bind orchelate to a particular ion or molecule, shielding its charge from thesurrounding environment, and thus facilitating its crossing of thehydrophobic interior of the lipid membrane. The second classificationis; channel formers that introduce a hydrophilic pore into the membrane,allowing molecules or metal ions to pass through while avoiding contactwith the hydrophobic interior of the membrane.

In conventional methods using ionophores, or other components capable oftransporting ions or loading of nanoparticles, the resultingnanoparticles comprise small amounts of the ion-transporter or ionophoreused in the loading procedure. The nanoparticles provided by the presentinvention are prepared without the use of an ion-transporter such as anionophore. Thus, the present invention relates to nanoparticlecompositions, which do not comprise ion-transporters or ionophores.

In another embodiment of the present invention, the nanoparticlecompositions as defined herein do not comprise any added ionophores.

Ion-transporters or ionophoric compounds which are not comprised in thenanoparticles of the present invention may be selected from the group of8-hydroxyquinoline (oxine); 8-hydroxyquinoline β-D-galactopyranoside;8-hydroxyquinoline β-D-glucopyranoside; 8-hydroxyquinoline glucuronide;8-hydroxyquinoline-5-sulfonic acid; 8-hydroxyquinoline-β-D-glucuronidesodium salt; 8-quinolinol hemisulfate salt; 8-quinolinol N-oxide;2-amino-8-quinolinol; 5,7-dibromo-8-hydroxyquinoline;5,7-dichloro-8-hydroxyquinoline; 5,7-diiodo-8-hydroxyquinoline;5,7-dimethyl-8-quinolinol; 5-amino-8-hydroxyquinoline dihydrochloride;5-chloro-8-quinolinol; 5-nitro-8-hydroxyquinoline;7-bromo-5-chloro-8-quinolinol; N-butyl-2,2′-imino-di(8-quinolinol);8-hydroxyquinoline benzoate; 2-benzyl-8-hydroxyquinoline;5-chloro-8-hydroxyquinoline hydrochloride; 2-methyl-8-quinolinol;5-chloro-7-iodo-8-quinolinol; 8-hydroxy-5-nitroquinoline;8-hydroxy-7-iodo-5-quinolinesulfonic acid;5,7-dichloro-8-hydroxy-2-methylquinoline, and other quinolines(1-azanaphthalene, 1-benzazine) consisting chemical compounds andderivatives thereof. In one embodiment the ionophoric compound isselected from the group consisting of: 8-hydroxyquinoline (oxine);8-hydroxyquinoline β-D-galactopyranoside; 8-hydroxyquinolineβ-D-glucopyranoside; 8-hydroxyquinoline glucuronide;8-hydroxyquinoline-5-sulfonic acid; 8-hydroxyquinoline-β-D-glucuronidesodium salt; 8-quinolinol hemisulfate salt; 8-quinolinol N-oxide;2-amino-8-quinolinol; 5,7-dibromo-8-hydroxyquinoline;5,7-dichloro-8-hydroxyquinoline; 5,7-diiodo-8-hydroxyquinoline;5,7-dimethyl-8-quinolinol; 5-amino-8-hydroxyquinoline dihydrochloride;5-chloro-8-quinolinol; 5-nitro-8-hydroxyquinoline;7-bromo-5-chloro-8-quinolinol; N-butyl-2,2′-imino-di(8-quinolinol);8-hydroxyquinoline benzoate; 2-benzyl-8-hydroxyquinoline;5-chloro-8-hydroxyquinoline hydrochloride; 2-methyl-8-quinolinol;5-chloro-7-iodo-8-quinolinol; 8-hydroxy-5-nitroquinoline;8-hydroxy-7-iodo-5-quinolinesulfonic acid;5,7-dichloro-8-hydroxy-2-methylquinoline, and other quinolines(1-azanaphthalene, 1-benzazine) consisting chemical compounds andderivatives thereof.

Ion-transporters or ionophoric compounds which are not comprised in thenanoparticles or used in the methods of the present invention mayadditionally be selected from the group consisting of2-hydroxyquinoline-4-carboxylic acid; 6-chloro-2-hydroxyquinoline;8-chloro-2-hydroxyquinoline; carbostyril 124; carbostyril 165;4,6-dimethyl-2-hydroxyquinoline; 4,8-dimethyl-2-hydroxyquinoline; orother 2-quinolinol compounds 8-hydroxyquinoline (oxine);8-hydroxyquinoline β-D-galactopyranoside; 8-hydroxyquinolineβ-D-glucopyranoside; 8-hydroxyquinoline glucuronide;8-hydroxyquinoline-5-sulfonic acid; 8-hydroxyquinoline-β-D-glucuronidesodium salt; 8-quinolinol hemisulfate salt; 8-quinolinol N-oxide;2-amino-8-quinolinol; 5,7-dibromo-8-hydroxyquinoline;5,7-dichloro-8-hydroxyquinoline; 5,7-diiodo-8-hydroxyquinoline;5,7-dimethyl-8-quinolinol; 5-amino-8-hydroxyquinoline dihydrochloride;5-chloro-8-quinolinol; 5-nitro-8-hydroxyquinoline;7-bromo-5-chloro-8-quinolinol; N-butyl-2,2′-imino-di(8-quinolinol);8-hydroxyquinoline benzoate; 2-benzyl-8-hydroxyquinoline;5-chloro-8-hydroxyquinoline hydrochloride; 2-methyl-8-quinolinol;5-chloro-7-iodo-8-quinolinol; 8-hydroxy-5-nitroquinoline;8-hydroxy-7-iodo-5-quinolinesulfonic acid;5,7-dichloro-8-hydroxy-2-methylquinoline, and other quinolines(1-azanaphthalene, 1-benzazine) consisting chemical compounds andderivatives thereof, [6S-[6.alpha.(2S*,3S*), 8.beta.(R*),9.beta.,11.alpha]]-5-(methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol2-yl)ethyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazolecarboxylicacid (also called A23187), HMPAO (hexamethyl propylene amine oxime,HYNIC (6-Hydrazinopyridine-3-carboxylic acid), BMEDA(N—N-bis(2-mercaptoethyl)-N′,N′-diethylethylenediamine), DISIDA(diisopropyl iminodiacetic acid, phthaldialdehyde and derivativesthereof, 2,4-dinitrophenol and derivatives thereof, di-benzo-18-crown-6and derivatives thereof, o-xylylenebis(N,N-diisobutyldithiocarbamate)and derivatives thereof, N,N,N′,N′-Tetracyclohexyl-2,2′-thiodiacetamideand derivates thereof,2-(1,4,8,11-Tetrathiacyclotetradec-6-yloxy)hexanoic acid,2-(3,6,10,13-Tetrathiacyclotetradec-1-oxy)hexanoic acid and derivatesthereof, N,N-bis(2-mercaptoethyl)-N′,N′-diethylethylenediamine andderivates thereof, beauvericin, enniatin, gramicidin, ionomycin,lasalocid, monesin, nigericin, nonactin, nystatin, salinomycin,valinomycin, pyridoxal isonicotinoyl hydrazone (PIH), salicylaldehydeisonicotinoyl hydrazone (SIH),1,4,7-trismercaptoethyl-1,4,7-triazacyclononane,N,N′,N″-tris(2-mercaptoethyl)-1,4,7-triazacyclononane, monensis, DP-b99,DP-109, BAPTA, pyridoxal isonicotinoyl hydrazone (PIH), alamethicin,di-2-pyridylketone thiosemicarbazone (HDpT), carbonyl cyanidem-chlorophenyl hydrazone (CCCP), lasalocid A (X-537A), 5-bromoderivative of lasalocid; cyclic depsipeptides; cyclic peptides: DECYL-2;N,N,N′,N′-tetrabutyl-3,6-dioxaoctanedi[thioamide]);N,N,N′,N′-tetracyclohexyl-3-oxa-pentanediamide;N,N-dicyclohexyl-N′,N′-dioctadecyl-diglycolic-diamide;N,N′-diheptyl-N,N′-dimethyl-1,-butanediamide;N,N″-octamethylene-bis[N′-heptyl-N′-methyl-malonamide;N,N-dioctadecyl-N′,N′-dipropyl-3,6-dioxaoctanediamide;N-[2-(1H-pyrrolyl-methyl)]-N′-(4-penten-3-on-2)-ethane-1,2-diamine(MRP20); and antifungal toxins; avenaciolide or derivatives of the abovementioned ionophores, as well as the ionophores described inWO2011/006510 and other ionophores described in the art.

pH gradient loadable agents i.e. agents with one or more ionisablemoieties such that the neutral form of the ionisable moiety allows themetal entities to cross the liposome membrane and conversion of themoiety to a charged form causes the metal entity to remain encapsulatedwithin the liposome are also regarded as ionophores according to thepresent invention. Ionisable moieties may comprise, but are not limitedto comprising, amine, carboxylic acid and hydroxyl groups. pH gradientloadable agents that load in response to an acidic interior may compriseionisable moieties that are charged in response to an acidic environmentwhereas drugs that load in response to a basic interior comprisemoieties that are charged in response to a basic environment. In thecase of a basic interior, ionisable moieties including but not limitedto carboxylic acid or hydroxyl groups may be utilized.

Interior PH

The interior pH of the nanoparticles according to the present inventioncan be controlled to lie in a specific range wherein the features of thenanoparticle are optimized.

In one embodiment of the present invention or the method of the presentinvention, the interior pH of the liposome composition is controlled,thus achieving a desired protonation state of the agent-entrappingcomponent and/or the ionophore, thereby securing efficient loading andentrapment of the radionuclide.

In a preferred embodiment of the present invention or the method of thepresent invention, the interior pH of the liposome composition iscontrolled, thus achieving a desired protonation state of theagent-entrapping component, thereby securing efficient loading andentrapment of the radionuclide.

In another embodiment of the disclosed method for producing ananoparticle composition loaded with a copper isotope, the interior pHis controlled during synthesis of the nanoparticles in such a way thatthe interior pH of the nanoparticles is within the range of 1 to 10,such as 1-2, for example 2-3, such as 3-4, for example 4-5, such as 5-6,for example 6-7, such as 7-8, for example 8-9, such as 9-10.

In a preferred embodiment of the present invention, the interior pH ofthe nanoparticles is in the range of 4 to 8.5, such as 4.0 to 4.5, forexample 4.5 to 5.0, such as 5.0 to 5.5 for example 5.5 to 6.0, such as6.0 to 6.5, for example 6.5 to 7.0, such as 7.0 to 7.5, for example 7.5to 8.0, such as 8.0 to 8.5.

In another embodiment of the present invention, the interior pH of thenanoparticles according to the present invention is optimized in orderto prolong the stability of the nanoparticles. Such improved stabilitycan for example lead to a longer shelf-life or a wider range of possiblestorage temperatures and thereby facilitate the use of thenanoparticles. The improved stability can be obtained, for examplebecause the interior pH leads to an increased stability of the vesicleforming components forming a vesicle, due to increased stability of theagent-entrapping component with or without the entrapped radionuclidesor due to improved stability of other features of the nanoparticles. Aninterior pH which is optimized for improved stability may be within therange of 1 to 10, such as 1-2, for example 2-3, such as 3-4, for example4-5, such as 5-6, for example 6-7, such as 7-8, for example 8-9, such as9-10.

In a preferred embodiment of the present invention, the interior pHwhich leads to an improved stability of the nanoparticles is in therange of 4 to 8.5, such as 4.0 to 4.5, for example 4.5 to 5.0, such as5.0 to 5.5 for example 5.5 to 6.0, such as 6.0 to 6.5, for example 6.5to 7.0, such as 7.0 to 7.5, for example 7.5 to 8.0, such as 8.0 to 8.5.

Osmotic Pressure

The creation of a small osmotic stress on the vesicle or nanoparticlemembrane is favourable in the loading of metal entity and/orradionuclide into the nanoparticles. Osmotic stress is a difference inosmotic pressure, i.e. an imbalance or difference between interior andexterior osmolarity. Presence of an osmotic stress facilitates thetransfer of smaller ions over the membrane, such as metal ions orradionuclide ions, while larger charged molecules such as chelatingagents remain trapped within the nanoparticles.

According to the present invention, the loading into liposomes can bemodulated by controlling the osmolarity of the aqueous solution that isencapsulated in the liposomes as well as the exterior solution duringmanufacture of the liposomes. The osmolarity (Osm) is a measure of theactivity of water (as a function of the chemical potential), which isgoverned by the presence of solutes in the aqueous solution, includingchelators or other osmotically active agents. Trans-membrane gradientsof osmolytes influences the state of the liposome and can either causethe liposome to be flaccid (Osm_(interior)<Osm_(exterior)) due to lossof water or to be tense due to uptake of water building up osmotictrans-membrane pressure (Osm_(interior)>Osm_(exterior)) and membranetension. In general, membrane tension will lead to membrane stretchingand thus thinning of the bilayer causing an increased permeability.Furthermore, membrane tension can cause formation of defects (transientpores), which also attributes to increased membrane permeability. It isthus expected that the membrane permeability increases with augmentedhyper-osmotic pressure (Osm_(interior)>Osm_(exterior)) leading to higherloading rate and loading efficiency. A too high osmotic pressure(tension) can also induce lysis of the liposomes and cause a gradualrelease of contents or mechanical failure of the liposome. For example,when 100 nm vesicles are placed in a hypo-osmotic solution with respectto the trapped intra-vesicular medium, it can result in an influx ofwater causing the vesicles to assume a spherical shape, and osmoticdifferentials of sufficient magnitude will produce membrane rupture thatresults in partial release of the intra-vesicular solutes. However, itis recognized that the presence of cholesterol in the membrane providemechanical stability thereby increasing the membrane lysis tensionresulting overall in a larger tolerance towards osmotic imbalance.

The intra-liposomal osmolarity can be determined by measuring theosmolarity of the solution used to hydrate lipid films during liposomepreparation, by using conventional methods in the art such as, but notlimited to the freeze-point method, which is commonly used in apparatusfor measuring osmolarity. The same method can be utilized for measuringthe osmolarity of the external liposomal buffer. Importantly, the bufferosmolarity is easily influenced by pH adjustment (using e.g. NaOH orHCl) during buffer preparation.

In a preferred embodiment of the present invention, the osmolarity ismeasured by use of the freeze point method.

Controlling the osmolarity can be used to create osmotic stress. Suchosmotic stress can be controlled by entrapping osmotic agents such assugars, salts, chelating agents, ions, peptides, proteins,pharmaceutical compounds, buffer molecules and/or solutes in thenanoparticles.

In one embodiment of the present invention, the osmolarity of theinterior of the nanoparticles is 40-800 mOsm/L, such as 40-100 mOsm/L,or such as 100-150 mOsm/L, or such as 150-200 mOsm/L, or such as 200-250mOsm/L, or such as 250-300 mOsm/L, or such as 300-350 mOsm/L, or such as350-400 mOsm/L, or such as 400-450 mOsm/L, or such as 450-500 mOsm/L, orsuch as 500-550 mOsm/L, or such as 550-600 mOsm/L, or such as 600-650mOsm/L, or such as 650-700 mOsm/L or such as 700-750 mOsm/L, or such as750-800 mOsm/L.

In another embodiment of the present invention, the osmolarity of theexterior of the nanoparticles is 40-800 mOsm/L, such as 40-100 mOsm/L,or such as 100-150 mOsm/L, or such as 150-200 mOsm/L, or such as 200-250mOsm/L, or such as 250-300 mOsm/L, or such as 300-350 mOsm/L, or such as350-400 mOsm/L, or such as 400-450 mOsm/L, or such as 450-500 mOsm/L, orsuch as 500-550 mOsm/L, or such as 550-600 mOsm/L, or such as 600-650mOsm/L, or such as 650-700 mOsm/L or such as 700-750 mOsm/L, or such as750-800 mOsm/L.

In one embodiment of the present invention, the difference in osmolaritybetween the interior of the nanoparticle and the exterior of thenanoparticle is 5-800 mOsm/L, such as 5-10 mOsm/L such as 10-20 mOsm/L,or such as 10-20 mOsm/L, or such as 20-20-30 mOsm/L, or such as 30-40mOsm/L, or such as 40-50 mOsm/L, or such as 50-60 mOsm/L, or such as60-70 mOsm/L, or such as 60-70 mOsm/L, or such as 70-80 mOsm/L, or suchas 80-90 mOsm/L, or such as 90-100 mOsm/L, or such as 100-150 mOsm/L, orsuch as 150-200 mOsm/L, or such as 200-250 mOsm/L, or such as 250-300mOsm/L, or such as 300-350 mOsm/L, or such as 350-400 mOsm/L, or such as400-450 mOsm/L, or such as 450-500 mOsm/L, or such as 500-550 mOsm/L, orsuch as 550-600 mOsm/L, or such as 600-650 mOsm/L, or such as 650-700mOsm/L, or such as 700-750 mOsm/L, or such as 750-800 mOsm/L.

In one particular embodiment of the present invention, the difference inosmolarity between the interior of the nanoparticle and the exterior ofthe nanoparticle is 10-100 mOsm/L.

Stability

The nanoparticles of the present invention have improved stability,which may be measured using different tests.

In one embodiment of the present invention, the stability of theradiolabeled nanoparticles is such that less than 20% leakage ofradioactivity is observed following a given time of incubation in bufferor human serum. Such leakage can be less than 20%, for example less than15% leakage, such as less than 12% leakage, for example less than 10%leakage, such as less than 8% leakage, for example less than 6% leakage,such as less than 4% leakage, for example less than 3% leakage, such asless than 2% leakage, for example less than 1% leakage as measured byconventional methods in the art, including a purification procedure suchas size exclusion chromatograpy (SEC), ion-exchange chromatography ordialysis. The amount of metal entity such as the radionuclide can bemeasured as radioactivity using a radioactivity detector or by measuringthe concentration of the metal entity using inductively coupled plasmamass spectrometry (ICP-MS), inductively coupled plasma atomic emissionspectroscopy (ICP-AES) or inductively coupled plasma optical emissionspectrometry (ICP-OES).

In one embodiment of the present invention, the stability of theradiolabeled nanoparticles is such that less than 20% leakage ofradioactivity is observed following 24 hours incubation in buffer orhuman serum at 37° C. followed by a purification procedure to separatethe radiolabeled nanoparticles from leaked radionuclide, for exampleless than 15% leakage, such as less than 12% leakage, for example lessthan 10% leakage, such as less than 8% leakage, for example less than 6%leakage, such as less than 4% leakage, for example less than 3% leakage,such as less than 2% leakage, for example less than 1% leakage. Saidpurification procedure comprises size exclusion chromatograpy (SEC),ion-exchange chromatography or dialysis. The amount of metal entity suchas the radionuclide is measured as radioactivity using a radioactivitydetector or by measuring the concentration of the metal entity usinginductively coupled plasma mass spectrometry (ICP-MS), inductivelycoupled plasma atomic emission spectroscopy (ICP-AES) or inductivelycoupled plasma optical emission spectrometry (ICP-OES).

Sizes of the Nanoparticles

Nanoparticles according to the present invention may vary in size. Thesize of the nanoparticles may be optimized for the use of thenanoparticle wherein the nanoparticle is administered to a subject, forexample for improving targeting of the particles to sites in the humanbody, or for improved monitoring of the nanoparticles inside the humanbody. The size may also be optimized for improved for stability of thenanoparticle or for improved or facilitated preparation of thenanoparticles.

In one embodiment, the nanoparticle composition of the present inventionhas a diameter in the range of 30 nm to 1000 nm; such as 30 nm to 300nm, such as 30 nm to 60 nm, for example 60 nm to 80 nm, such as 80 nm to100 nm, for example 100 nm to 120 nm, such as 120 nm to 150 nm, forexample 150 nm to 180 nm, such as 180 nm to 210 nm, for example, 210 nmto 240 nm, such as 240 nm to 270 nm for example 270 nm to 300 nm, orsuch as 300 nm to 600 nm, such as 300 nm to 400 nm, or such as 400 nm to500 nm, or such as 500 nm to 600 nm, or such as 600 nm to 1000 nm, suchas 600 nm to 700 nm, or such as 700 nm to 800 nm, or such as 800 nm to900 nm, or such as 900 nm to 1000 nm.

In one embodiment, the disclosed method for producing a nanoparticleloaded with radionuclides results in nanoparticles which has a diameterin the range of 30 nm to 1000 nm; such as 30 nm to 300 nm, such as 30 nmto 60 nm, for example 60 nm to 80 nm, such as 80 nm to 100 nm, forexample 100 nm to 120 nm, such as 120 nm to 150 nm, for example 150 nmto 180 nm, such as 180 nm to 210 nm, for example, 210 nm to 240 nm, suchas 240 nm to 270 nm for example 270 nm to 300 nm, or such as 300 nm to600 nm, such as 300 nm to 400 nm, or such as 400 nm to 500 nm, or suchas 500 nm to 600 nm, or such as 600 nm to 1000 nm, such as 600 nm to 700nm, or such as 700 nm to 800 nm, or such as 800 nm to 900 nm, or such as900 nm to 1000 nm.

In one embodiment, the disclosed method for producing a nanoparticleloaded with copper results in nanoparticles which has a diameter in therange of 30 nm to 1000 nm; such as 30 nm to 300 nm, such as 30 nm to 60nm, for example 60 nm to 80 nm, such as 80 nm to 100 nm, for example 100nm to 120 nm, such as 120 nm to 150 nm, for example 150 nm to 180 nm,such as 180 nm to 210 nm, for example, 210 nm to 240 nm, such as 240 nmto 270 nm for example 270 nm to 300 nm, or such as 300 nm to 600 nm,such as 300 nm to 400 nm, or such as 400 nm to 500 nm, or such as 500 nmto 600 nm, or such as 600 nm to 1000 nm, such as 600 nm to 700 nm, orsuch as 700 nm to 800 nm, or such as 800 nm to 900 nm, or such as 900 nmto 1000 nm.

In a preferred embodiment, the nanoparticle composition of the presentinvention has a diameter in the range of 30 nm to 300 nm; such as 30 nmto 60 nm, for example 60 nm to 80 nm, such as 80 nm to 100 nm, forexample 100 nm to 120 nm, such as 120 nm to 150 nm, for example 150 nmto 180 nm, such as 180 nm to 210 nm, for example, 210 nm to 240 nm, suchas 240 nm to 270 nm for example 270 nm to 300 nm.

In a preferred embodiment, the disclosed method for producing ananoparticle loaded with radionuclides results in nanoparticles whichhas a diameter in the range of 30 nm to 300 nm; such as 30 nm to 60 nm,for example 60 nm to 80 nm, such as 80 nm to 100 nm, for example 100 nmto 120 nm, such as 120 nm to 150 nm, for example 150 nm to 180 nm, suchas 180 nm to 210 nm, for example, 210 nm to 240 nm, such as 240 nm to270 nm for example 270 nm to 300 nm.

In a preferred embodiment, the disclosed method for producing ananoparticle loaded with copper results in nanoparticles which has adiameter in the range of 30 nm to 300 nm; such as 30 nm to 60 nm, forexample 60 nm to 80 nm, such as 80 nm to 100 nm, for example 100 nm to120 nm, such as 120 nm to 150 nm, for example 150 nm to 180 nm, such as180 nm to 210 nm, for example, 210 nm to 240 nm, such as 240 nm to 270nm for example 270 nm to 300 nm.

Methods for Preparation

The present invention provides methods for preparation of nanoparticlecompositions as described herein comprising a vesicle forming component,an agent-entrapping component enclosed by the vesicle forming component,and an entrapped metal entity within the nanoparticle composition.

Such methods for preparation of nanoparticles according to the presentinvention comprise the following steps:

-   -   a. Providing a nanoparticle composition comprising a vesicle        forming component and an agent-entrapping component enclosed by        said vesicle forming component;    -   b. Entrapping the metal entities within the interior of the        nanoparticle composition by incubation of the nanoparticle        composition in a solution comprising the metal entity at a        temperature higher than 22° C.

Preferred methods for preparation of nanoparticles according to thepresent invention which do not involve the use of ionophore for loading,comprise the following steps:

-   -   a. Providing a nanoparticle composition comprising a vesicle        forming component and an agent-entrapping component enclosed by        said vesicle forming component;    -   b. Entrapping the metal entities within the interior of the        nanoparticle composition by enabling transfer of cation metal        entities across the membrane of the vesicle forming component by        incubation of the nanoparticle composition in a solution        comprising the metal entity.

In one embodiment, the methods according to the present invention, theincubation temperature for loading of the nanoparticles is higher than22° C., such as higher than 30° C., such as higher than 35° C., such ashigher than 40° C., such as higher than 45° C., such as higher than 50°C., such as higher than 55° C., such as higher than 60° C., such ashigher than 65° C., such as higher than 70° C., such as higher than 75°C.

In another embodiment, the methods according to the present invention,the incubation temperature for loading of the nanoparticles is higherthan 10° C., such as higher than 15° C., such as higher than 22° C.,such as higher than 30° C., such as higher than 35° C., such as higherthan 40° C., such as higher than 45° C., such as higher than 50° C.,such as higher than 55° C., such as higher than 60° C., such as higherthan 65° C., such as higher than 70° C., such as higher than 75° C.

In the methods according to the present invention, the incubationtemperature for loading of the nanoparticles is lower than a criticaltemperature where upon the nanoparticles will degrade. Thus according tothe present invention, the incubation temperature for loading of thenanoparticles is lower than 100° C., such as lower than 90° C., such aslower than 85° C., such as lower than 80° C.

In yet another embodiment of the present invention, incubationtemperature for loading of the nanoparticles is between 22° C. to 80°C., such as 22° C. to 30° C., or in the range of 30° C. to 80° C., suchas in the range of 30° C. to 40° C., such as 30° C. to 35° C., or suchas 35° C. to 40° C., or in the range of 40° C. to 80° C., such as 40° C.to 45° C., or such as 45° C. to 50° C., including the range of 50° C. to80° C., such as 50° C. to 55° C., or such as 55° C. to 60° C., or suchas 60° C. to 65° C., or such as 65° C. to 70° C., or such as 70° C. to75° C., or such as 75° C. to 80° C., wherein a range of 30° C. to 80° C.is preferred and a range of 40° C. to 80° C. is more preferred.

In yet another embodiment of the present invention, incubationtemperature for loading of the nanoparticles is between 10° C. to 80°C., such as 15° C. to 80° C., or such as 15° C. to 22° C., or in therange of 22° C. to 80° C., such as 22° C. to 30° C., such as in therange of 30° C. to 80° C., such as in the range of 30° C. to 40° C.,such as 30° C. to 35° C., or such as 35° C. to 40° C., or in the rangeof 40° C. to 80° C., such as 40° C. to 45° C., or such as 45° C. to 50°C., including the range of 50° C. to 80° C., such as 50° C. to 55° C.,or such as 55° C. to 60° C., or such as 60° C. to 65° C., or such as 65°C. to 70° C., or such as 70° C. to 75° C., or such as 75° C. to 80° C.,wherein a range of 30° C. to 80° C. is preferred and a range of 40° C.to 80° C. is more preferred.

The methods of the present invention allows for a faster loading of thenanoparticles than what is expected from mere diffusion of the metalentities/radionuclides into the nanoparticles.

Thus, in one embodiment of the present invention, the incubation forloading of the nanoparticles can be performed in a time period which isless than 48 hours, such as 36-48 hours, or such as 24-36 hours, or suchas 18-24 hours, or such as 16-18 hours, or such as 14-16 hours, or suchas 12-14 hours, or such as 10-12 hours, or such as 8-10 hours, or suchas 6-8 hours, or such as 4-6 hours, or such as 2-4 hours, or such as 1-2hours, or such as 30 min to 60 min, or 5 min to 30 min, or 1 min to 5min.

The incubation time according to the present invention is a time periodshorter than 48 hours, such as between 0 minutes to 360 minutes, such asbetween 1 minutes to 240 minutes preferably between 1 minutes to 120minutes (including 3 minutes to 120 minutes and 5 minutes to 120minutes) and more preferably between 1 minutes to 60 minutes, such as 5minutes to 60 minutes.

The methods of the present invention may comprise one or more stepswherein an osmotic stress as defined herein is created in thenanoparticles. The inventors have found that a difference in theosmolarity of the interior of the nanoparticle compared to the exteriorof the nanoparticle improves the loading of the nanoparticles. Saidosmotic stress can be created by ensuring that there is an imbalancebetween the interior ion concentration of the nanoparticles compared tothe exterior ion concentration, thus a difference in the osmoticpressure over the membrane of the vesicle.

Such osmotic stress or osmotic pressure can be controlled by anentrapped osmotic agent such as salts, sugars, ions, chelates, peptides,proteins, pharmaceutical compounds, buffer molecules, and/or othersolutes.

In one embodiment of the present invention, the osmotic pressure of themembrane is obtained by controlling the osmolarity of the interior ofthe nanoparticle by preparing the nanoparticle composition in step a)using a solution which has an osmolarity for enhancing the loading,wherein said solution comprises one or more osmotic agents as definedherein.

In another embodiment of the present invention, the osmotic pressure ofthe membrane is obtained by controlling the osmolarity of the exteriorof the nanoparticle by incubating the nanoparticle composition in stepb) using a solution which has an osmolarity for enhancing the loading,wherein said solution comprises one or more osmotic agents as definedherein.

Thus in one embodiment of the present invention, the difference inosmolarity of the interior of the nanoparticle compared to theincubation solution (exterior of the nanoparticle) is 5-800 mOsm/L atthe starting point of the incubation.

The methods of the present invention ensure that a high amount of theradionuclides used in preparation will be entrapped within thenanoparticle. In one embodiment of the present invention or the methodof the present invention, the efficiency of loading is higher than 90%when assayed using size exclusion chromatography (SEC, described inexamples), ion-exchange chromatography or dialysis. In anotherembodiment of the present method the efficiency of loading is higherthan 35%, for example higher than 40%, such as higher than 50%, forexample higher than 60%, such as higher than 65%, for example higherthan 70%, such as higher than 75%, for example higher than 80%, such ashigher than 85%, for example higher than 90%, such as higher than 95%,or such as higher than 96%, or such as higher than 97%, or such ashigher than 98%, or such as higher than 99%.

In one embodiment of the present invention, the loading efficiency whenusing incubation times of 1 minutes to 240 minutes is in the range of10% to 100%, preferably in the range of 80% to 100% and more preferablyin the range of 90% to 100%, such as for example in the range of95%-100% (including 95% to 99.9%, such as 95%-99%).

Thus in one embodiment of the present invention, the incubationtemperature for loading of nanoparticles is in the range of 30° C. to80° C. and the loading efficiency when using incubation times of 1 to240 minutes is in the range of 10% to 100% preferably in the range of80% to 100% and more preferably in the range of 90% to 100%, such as forexample in the range of 95%-100% (including 95% to 99.9%, such as95%-99%).

In a preferred embodiment of the present invention, the incubationtemperature for loading of nanoparticles is in the range of 30° C. to80° C. and the loading efficiency when using incubation times of 1minutes to 60 minutes is in the range of 10% to 100%, preferably in therange of 80% to 100%, such as 90%-100%, and more preferably in the rangeof 95% to 100%, such as 95% to 99.9%, or such as 95%-99%).

The methods of the present invention may comprise a step wherein theloaded nanoparticles are purified from the incubation solution asmentioned in step b). Thus, in one embodiment of the invention or thedisclosed method of the invention, the generated nanoparticles loadedwith radionuclides are purified using SEC, ion-exchange chromatographyor dialysis.

In a preferred embodiment of the invention or the disclosed method ofthe invention, the generated nanoparticles loaded with copper arepurified using SEC, ion-exchange chromatography or dialysis.

In one embodiment of the disclosed method, the size of the nanoparticlecompositions remains essentially unaltered following loading of saidnanoparticles with copper. In another embodiment of the disclosedmethod, the size of the nanoparticle compositions is altered less than20% following loading of the nanoparticles with copper isotopes, forexample less than 17%, such as less than 14%, for example less than 11%,such as less than 8%, for example less than 5%, such as less than 2%,for example less than 1%.

In one embodiment of the disclosed method, the size of the nanoparticlecompositions remains essentially unaltered following loading of saidnanoparticles with a radionuclide. In another embodiment of thedisclosed method, the size of the nanoparticle compositions is alteredless than 20% following loading of the nanoparticles with aradionuclide, for example less than 17%, such as less than 14%, forexample less than 11%, such as less than 8%, for example less than 5%,such as less than 2%, for example less than 1%.

In one embodiment of the disclosed method, the zeta potential is alteredless than 20% following loading of the nanoparticles with copperisotopes. In another embodiment of the disclosed method, the zetapotential is altered less than 18% following loading of thenanoparticles with copper isotopes, for example less than 16%, such asless than 14%, for example less than 12%, such as less than 10%.

In one embodiment of the disclosed method, the zeta potential is alteredless than 20% following loading of the nanoparticles with aradionuclide. In another embodiment of the disclosed method, the zetapotential is altered less than 18% following loading of thenanoparticles with a radionuclide, for example less than 16%, such asless than 14%, for example less than 12%, such as less than 10%.

In a further embodiment the method for preparing nanoparticlecompositions encompass controlling the liposome interior pH in the formof protonating or deprotonating the agent-entrapping component, therebyinducing effective loading of the radionuclide.

The described method for preparing nanoparticle compositions may furthercomprise a step wherein a moiety is attached or associated to theexternal layer of the nanoparticle which is targeted for a cancerousdisease, and in general, pathological conditions associated with leakyblood vessels. In another embodiment of the present invention, methodfor preparing nanoparticle compositions further comprises step wherein acompound with intracellular targeting properties, such as nuclearlocalization sequence peptide (NLS peptide), is conjugated to theagent-entrapping component, and/or entrapped within the nanoparticlecomposition.

A method for preparation of the disclosed nanoparticle composition mayfurther comprise a step of measuring and/or detecting the amount ofradiation emitted from the radionuclide entrapped within thenanoparticle composition.

The methods provided by the present invention do not include the use ofan ion-transporter such as an ionophore. Thus nanoparticles prepared bythe methods of the present invention do not comprise ion-transporters,lipophilic chelators or ionophores.

Kit of Parts

The present invention provides kit of parts for preparation of thenanoparticles.

According to the present invention, such a kit of parts may comprise:

-   -   a. A nanoparticle composition comprising i) a vesicle forming        component, and ii) an agent-entrapping component enclosed by the        vesicle forming component; and    -   b. A composition containing a metal entity for loading into the        nanoparticle,        wherein all the components are as described herein.

In one embodiment, the composition containing a metal entity comprises aradionuclide.

The metal entity or radionuclide is either in storage or delivered fromthe manufacturer depending on the characteristics of the particularradionuclide. The radionuclide may be delivered in the form of a(lyophilized) salt or an aqueous solution or may be synthesized on thepremises using existing production facilities and starting materials.Before administration of the radionuclide-containing nanoparticles,parts a, and b of the kit are mixed, and incubated at a temperaturehigher than 22° C. for a given time period, wherein the incubationtemperature and time period are as defined herein.

The efficiency of encapsulation is then tested, preferably using thesimple test procedure supplied with the kit. Following test ofencapsulation, the drug is administered to the patient.

According to the present invention, a kit of parts may also comprise:

A mixture of a nanoparticle composition comprising a) a vesicle formingcomponent, and b) an agent-entrapping component enclosed by the vesicleforming component; and c) composition containing a metal entity forloading into the nanoparticle, wherein all the components are asdescribed in the present application. Before administration of theradionuclide-containing nanoparticles the mixture of parts a, b and c isincubated at a temperature higher than 22° C. for a given time period,wherein the incubation temperature and time period are as definedherein.

If the metal entity comprises a radionuclide e.g. the positron emitter⁶⁴Cu, said radionuclide is delivered directly from a cyclotron facilityto the venue of treatment or diagnosis immediately prior to use, in theform of a (lyophilized) salt or an aqueous solution. Beforeadministration of the radionuclide-containing nanoparticles, parts a andb of the kit are mixed and the efficiency of encapsulation is tested,preferably using the simple test procedure supplied with the kit.Following administration the patient may receive a PET- or a SPECT scan.Optimal visualization may be achieved 4-48 hours after administration.

In another embodiment of the present invention, the patient may besubject to magnetic resonance imaging (MRI) following administration ofthe nanoparticle compositions as mentioned herein. Such MRI may or maynot be combined with PET or SPECT scanning according to the presentinvention.

Thus, according to the present invention, a kit of parts may comprise:

-   -   a. A nanoparticle composition comprising i) a vesicle forming        component ii) an agent-entrapping component enclosed by the        vesicle forming component and iii) a metal entity useful for        MRI; and    -   b. A composition containing one or more metal entities for        loading into the nanoparticle,        wherein all the components are as described herein,        or, a kit of parts may comprise:    -   a. A nanoparticle composition comprising i) a vesicle forming        component and ii) an agent-entrapping component enclosed by the        vesicle forming component; and    -   b. A composition comprising one or more metal entities for        loading into the nanoparticle,        wherein all the components are as described herein.

In one embodiment of the present invention, the kit of parts comprise acombination of radionuclides useful for combined positron emissiontomography (PET) imaging and radiation therapy, for example tworadionuclides such as ⁶⁴Cu and ¹⁷⁷Lu, or such as ⁶⁴Cu and ⁶⁷Cu, or suchas ⁶¹Cu and ⁶⁷Cu, or such as ⁶⁴Cu and ⁹⁰Y, or such as ⁶⁴Cu and ¹¹⁹5 b,or such as ⁶⁴Cu and ²²⁵Ac, or such as ⁶⁴Cu and ¹⁸⁸Re, or such as ⁶⁴Cuand ¹⁸⁶Re, or such as ⁶⁴Cu and ²¹¹At.

In a preferred embodiment of the present invention, the kit of partscomprise a combination of radionuclides useful for combined positronemission tomography (PET) imaging and radiation therapy, such as ⁶⁴Cuand 177Lu.

In another embodiment of the present invention, said kit of parts is forpreparation of nanoparticles comprising radionuclides such as forexample isotopes of Copper (⁶¹Cu, ⁶⁴Cu, and ⁶⁷Cu), wherein said isotopesmay or may not be part of the kit of parts. In such an embodiment of thepresent invention, such a kit of parts may comprise: A nanoparticlecomposition comprising i) a vesicle forming component, and ii) anagent-entrapping component enclosed by the vesicle forming component. Ina further embodiment of the present invention, the kit of parts furthercomprises an incubation buffer for loading of the metal entities intothe nanoparticles.

In a preferred embodiment, any of the kit of parts further comprises atest procedure to assess the efficiency of encapsulation.

The kits of parts for preparation of nanoparticles according to thepresent invention, do not include an ion-transporter such as anionophore.

Methods for Treatment or Diagnosis

The nanoparticles of the present invention can be used for diagnosis,monitoring or treatment of diseases or conditions associated with leakyblood vessels in an animal subject in need, for example a mammal inneed, such as a human being in need.

Leaky blood vessels are often associated with angiogenesis or neoplasticgrowth of tissue. Cancer is an example of a disease characterized byleaky blood vessels. Inflammation is another example of a conditionsassociated with leaky blood vessels.

As mentioned herein, cancer is a disease characterized by leaky bloodvessels, and the present invention relates to treatment, monitoring ordiagnosis of cancerous diseases associated with malignant neoplasia suchas malignant neoplasm of lip, mouth or throat, such as malignantneoplasm of the tongue, the base of tongue, gum, floor of mouth, palate,parotid gland, major salivary glands, tonsil, oropharynx, nasopharynx,piriform sinus, hypopharynx or other parts of lip, mouth or throat ormalignant neoplasms of digestive organs such as malignant neoplasms ofoesophagus, stomach, small intestine, colon, rectosigmoid junction,rectum, anus and anal canal, liver and intrahepatic bile ducts,gallbladder, other parts of biliary tract, pancreas and spleen,malignant neoplasms of respiratory and intrathoracic organs such asmalignant neoplasms of the nasal cavity and middle ear, accessorysinuses, larynx, trachea, bronchus and lung, thymus, heart, mediastinumand pleura, malignant neoplasms of bone and articular cartilage such asmalignant neoplasm of bone and articular cartilage of limbs, bone andarticular cartilage, malignant melanoma of skin, sebaceous glands andsweat glands, malignant neoplasms of mesothelial and soft tissue such asmalignant neoplasm of mesothelioma, Kaposi's sarcoma, malignant neoplasmof peripheral nerves and autonomic nervous system, malignant neoplasm ofretroperitoneum and peritoneum, malignant neoplasm of connective andsoft tissue such as blood vessels, bursa, cartilage, fascia, fat,ligament, lymphatic vessel, muscle, synovia, tendon, head, face andneck, abdomen, pelvis or overlapping lesions of connective and softtissue, malignant neoplasm of breast or female genital organs such asmalignant neoplasms of vulva, vagina, cervix uteri, corpus uteri,uterus, ovary, Fallopian tube, placenta or malignant neoplasms of malegenital organs such as malignant neoplasms of penis, prostate, testis,malignant neoplasms of the urinary tract, such as malignant neoplasms ofkidney, renal pelvis, ureter, bladder, urethra or other urinary organs,malignant neoplasms of eye, brain and other parts of central nervoussystem such as malignant neoplasm of eye and adnexa, meninges, brain,spinal cord, cranial nerves and other parts of central nervous system,malignant neoplasms of thyroid and other endocrine glands such asmalignant neoplasm of the thyroid gland, adrenal gland, parathyroidgland, pituitary gland, craniopharyngeal duct, pineal gland, carotidbody, aortic body and other paraganglia, malignant neoplasms of head,face and neck, thorax, abdomen and pelvis, secondary malignant neoplasmof lymph nodes, respiratory and digestive organs, kidney and renalpelvis, bladder and other and urinary organs, secondary malignantneoplasms of skin, brain, cerebral meninges, or other parts of nervoussystem, bone and bone marrow, ovary, adrenal gland, malignant neoplasmsof lymphoid, haematopoietic and related tissue such as Hodgkin'sdisease, follicular non-Hodgkin's lymphoma, diffuse non-Hodgkin'slymphoma, peripheral and cutaneous T-cell lymphomas, non-Hodgkin'slymphoma, lymphosarcoma, malignant immunoproliferative diseases such asWaldenström's macroglobulinaemia, alpha heavy chain disease, gamma heavychain disease, immunoproliferative small intestinal disease, multiplemyeloma and malignant plasma cell neoplasms such as plasma cellleukaemia, plasmacytoma, solitary myeloma, lymphoid leukaemia such asacute lymphoblastic leukaemia, myeloid leukaemia, monocytic leukaemia,blast cell leukaemia, stem cell leukaemia, and other and unspecifiedmalignant neoplasms of lymphoid, haematopoietic and related tissue suchas Letterer-Siwe disease, malignant histiocytosis, malignant mast celltumour, true histiocytic lymphoma or other types of malignant neoplasia.

According to the present invention, a disease associated with leakyblood vessels also may be carcinoma in situ of oral cavity, oesophagus,stomach, digestive organs, middle ear and respiratory system, melanomain situ, carcinoma in situ of skin, carcinoma in situ of breast,carcinoma in situ of female or male genitals, carcinoma in situ ofbladder, urinary organs or eye, thyroid and other endocrine glands, orother types of carcinoma in situ.

The nanoparticles or vesicles of the present invention are preferablyfor administration to a subject such as a human being.

According to the present invention, the nanoparticles may beadministered to a subject in need in a manner which ensures the deliveryof the nanoparticles to tissues comprising leaky blood vessels. Suchadministration may ensure that the nanoparticles are brought intocirculation in the blood or the lymph.

In one embodiment of the present invention, the nanoparticles are usedfor intravenous administration.

In another embodiment of the present invention, the nanoparticles areused for oral administration.

The vesicles or nanoparticles according to the present invention may beused for one or more types of imaging. Such imaging may or be part of amethod for treating, monitoring or diagnosis of a disease, monitoringefficiency of treatment by chemotherapy or radiotherapy or conditionassociated with leaky blood vessels. Imaging according to the presentinvention comprises x-ray imaging, computed tomography (CT) imaging,magnetic resonance imaging (MRI), positron emission tomography (PET)imaging, single photon emission computed tomography (SPECT) imaging ornuclear scintigraphy imaging.

In one embodiment of the present invention, a method is provided formonitoring, monitoring treatment efficiency, diagnosis or treatment in asubject in need which comprises:

-   -   a. Providing a nanoparticle composition comprising a vesicle        forming component, an agent-entrapping component, and one or        more entrapped metal entities.    -   b. Administering the nanoparticle composition to a subject in        need.

In another embodiment of the present invention, a method is provided formonitoring, monitoring treatment efficiency, diagnosis or treatment in asubject in need which comprises:

-   -   a. Providing a nanoparticle composition comprising a vesicle        forming component, an agent-entrapping component, and one or        more radionuclides entrapped within the nanoparticle.    -   b. Administering the nanoparticle composition to a subject by        intravenous administration    -   c. Measuring the amount of radiation emitted from the        radionuclides within the liposome composition after a given        incubation time.        or    -   a. Providing a nanoparticle composition comprising a vesicle        forming component, an agent-entrapping component, and one or        more metal entities entrapped within the nanoparticle.    -   b. Administering the nanoparticle composition to a subject by        intravenous administration    -   c. Using conventional imaging methods for measuring the presence        and/or location of the metal entities in said subject.

In one embodiment of the present invention, a method for monitoring,monitoring treatment efficiency, diagnosis or treatment of cancer isprovided which comprises:

-   -   a. Providing a nanoparticle composition comprising a vesicle        forming component, an agent-entrapping component, and a        radiolabeled agent comprising one or more radionuclides of the        copper isotopes ⁶¹Cu, ⁶⁴Cu and ⁶⁷Cu which may be Cu(II) cations        or Cu(I) cations.    -   b. Administering the nanoparticle composition to a subject by        intravenous administration    -   c. Measuring the amount of radiation emitted from the        radionuclide within the liposome composition after a given        incubation time.

EXAMPLES Example I Improved Loading of ⁶⁴Cu and/or ¹⁷⁷Lu into LiposomesComprising a Chelating Agent

Preparation of Liposome Composition Containing Chelating-Agent:

Chelating-agent (DOTA) was trapped within the liposomes consisting of1,2-disteraroyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (CHOL)and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000(DSPE-PEG-2000) in the molar ratio 50:40:10 using standard thin-filmhydration and repeated extrusions. Briefly, the lipids were mixed inchloroform and dried to a lipid-film under a gentle stream of nitrogen.Organic solvent residues were removed under reduced pressure overnight.The lipid-film was dispersed by adding an aqueous solution—a HEPESbuffer (10 mM, 150 mM NaCl, pH 7.4) containing the chelating-agent,DOTA, adjusted to either pH 4.0 or pH 7.4 with a concentration of 10 mMand the osmolarity was measured to be 325 mOsm/L. The solution was thenhydrated at 65° C. for 60 min. Multi-lamellar vesicles (MLVs) were sizedto large unilamellar vesicles (LUVs) by multiple extrusions through 100nm polycarbonate filters using a mini-extruder. Unentrappedchelating-agent was removed by size exclusion chromatography (SEC) on aSephadex G-25 packed 1×25 cm column eluted with a HEPES buffer (10 mM,150 mM NaCl, pH 7.4, 310 mOsm/L).

Loading Liposomes with Radionuclide:

The suspension of liposomes prepared as described in the section above,was added to a dried vial containing a radionuclide such a ⁶⁴Cu and/or¹⁷⁷Lu. The suspension was incubated for 60 min at 50-55° C. Radionuclideloading efficiency was greater than 90% for ⁶⁴Cu and greater then 80%for ¹⁷⁷Lu. The separation of ⁶⁴Cu-Liposomes and free un-entrapped ⁶⁴Cuwith size exclusion chromatography (SEC) using Sephadex G-25 column isshown in FIG. 1. The separation of ¹⁷⁷Lu-Liposomes and free un-entrapped¹⁷⁷Lu with size exclusion chromatography (SEC) using Sephadex G-25column is shown in FIG. 2.

The loading efficiency of ⁶⁴Cu as function of temperature is shown inFIG. 3, and compared to ionophoric loading using 2-hydroxyquinoline(2HQ). The loading efficiency of ⁶⁴Cu into liposome compositions whenusing the ionophore 2HQ was slightly increasing as function oftemperature with a maximum loading efficiency (92.4%±1.4%) at 50-55° C.In contrast the loading efficiency of ⁶⁴Cu into liposome compositionswithout using an ionophore was increasing with increasing temperaturereaching a higher loading efficiency (96.7%±1.0%) at 50-55° C. comparedto the method with ionophore (2HQ).

Storage Stability at 37° C. for 24 h of ⁶⁴Cu-Liposome with a LiposomalInterior pH 4.0:

A purified 500 uL ⁶⁴Cu-Liposome solution was incubated at 37° C. for 24h, and the stability of the ⁶⁴Cu-Liposome were assayed by separatingfree ⁶⁴Cu from ⁶⁴Cu-Liposome by size exclusion chromatography (SEC) on aSephadex G-25 column. The elution profile was monitored on an in lineradioactivity detector. The ⁶⁴Cu-Liposomes containing 10 mM DOTA (pH4.0) retained more than 95% of the total radioactivity. The radionuclidebinds preferably to DOTA encapsulated in the interior of the liposome,due to its strong affinity thereto, allowing the entrapment of theradionuclide.

Storage Stability at 37° C. for 24 h of ⁶⁴Cu-Liposome with a LiposomalInterior pH 7.4:

A purified 500 uL ⁶⁴Cu-Liposome solution was incubated at 37° C. for 24h, and the stability of the ⁶⁴Cu-liposome were assayed by separatingfree ⁶⁴Cu from ⁶⁴Cu-Liposome by size exclusion chromatography (SEC) on aSephadex G-25 column. The elution profile was monitored on an in lineradioactivity detector. The ⁶⁴Cu-Liposomes containing 10 mM DOTA (pH7.4) retained more than 95% of the total radioactivity. The radionuclidebinds preferably to DOTA encapsulated in the interior of the liposome,due to its strong affinity thereto, allowing the entrapment of theradionuclide.

The disclosed method of producing nanoparticle compositions loaded withradionuclides is a fast and easy preparation of a novel PET imagingagents. The fast preparation is important due to the short half-life ofthe positron-emitter, ⁶⁴Cu, and ideal in manufacturing the product. Themethod is very robust and consistently gives high loading efficiencies(>95%) using liposome composition containing a chelating-agent, acontrolled osmotic pressure on the inside of the liposomes, and with apH ranging from 4 to 8. It is an advantage of the disclosed method thatthere are no carriers such as lipophilic ionophores used to load theradionuclides into the liposomes. The disclosed method of preparingnanoparticles containing radionuclides produces nanoparticlesretaining >95% of the entrapped radionuclides, which is a necessity forthe utilization of these nanoparticle compositions as imaging andtherapeutic agents.

Example II Preparation of Liposome Composition ContainingChelating-Agent for Cu(II)-Loading

The loading of non-radioactive Cu²⁺ into chelator-containing liposomeswas tested, and evaluated by using an ion Cu(II)-selective electrode.The electrode converts the activity of Cu²⁺ dissolved in a solution intoan electrical potential, which is measured by a voltmeter or pH meter.Thus the Cu(II)-selective electrode responds to un-complexed Cu²⁺ ionactivity. The sensing part of the electrode is made as an ion-specificmembrane, along with a reference electrode.

Chelating-agent (DOTA) was trapped within the liposomes consisting ofDSPC, cholesterol and DSPE-PEG-2000 in the molar ratio 50:40:10 usingstandard thin-film hydration and repeated extrusions. Briefly, thelipids were mixed in chloroform and dried to a lipid-film under a gentlestream of nitrogen. Organic solvent residues were removed under reducedpressure overnight. The lipid-film was dispersed by adding an aqueoussolution—a HEPES buffer (10 mM, 150 mM NaNO₃, pH 6.85) containing thechelating-agent, DOTA, adjusted to pH 4.0. The solution was thenhydrated at 65° C. for 60 min. Multi-lamellar vesicles (MLVs) were sizedto large unilamellar vesicles (LUVs) by multiple extrusions through 100nm polycarbonate filters using a mini-extruder. Unentrappedchelating-agent was removed by size exclusion chromatography (SEC) on aSephadex G-25 packed 1×25 cm column eluted with a HEPES buffer (10 mM,150 mM NaNO₃, pH 6.85, 310 mOsm/L).

Loading Liposomes with Cu(II):

A sequence of Cu(NO₃)₂ standard solutions were prepared and measuredusing a Cu(II)-selective electrode (FIG. 4). The Cu(II)-selectiveelectrode responds to uncomplexed copper ion activity. Cu(NO₃)₂ wasadded to the liposomes (final concentration of 25 ppm) and theCu(II)-electrode response was measured to 141 mV (FIG. 4) correspondingto 18.1 ppm uncomplexed Cu(II). The liposome suspension was incubatedfor 60 min at 50-55° C. for loading Cu(II) into the liposomecompositions, giving a Cu(II)-electrode response of 94 mV correspondingto 1.2 ppm Cu(II). The blank (background) measurement (10 mM HEPESbuffer, 150 mM NaNO₃, pH 6.85) without Cu(II) added gave aCu(II)-electrode response of 104 mV corresponding to 2.2 ppm Cu(II).

To calculate the loading efficiency the following equation (4) was used:

$\begin{matrix}{{{\left( {1 - \frac{1.2\mspace{14mu} {ppm}}{25\mspace{14mu} {ppm}}} \right) \cdot 100}\%} > {95\%}} & \left( {{equation}\mspace{14mu} 4} \right)\end{matrix}$

These results strongly indicates a very high loading efficiency (>95%)of Cu(II) into the liposomes compositions (FIG. 4), when using thedisclosed method.

Example III

To test whether the ionophore free loading method was limited todivalent cations, the two radioactive trivalent cations, ¹⁷⁷Lu³⁺ and¹¹¹In³⁺, were tested. Loading of the radioactive pertechnetate(^(99m)Tc), was also tested. ^(99m)Tc is an oxoanion with the chemicalformula TcO₄ ⁻. Successful loading of both ¹⁷⁷Lu³⁺ and ¹¹¹In³⁺ in intochelator-containing liposomes without using ionophores was obtained. Incontrast no loading was observed of ^(99m)TcO₄ ⁻ (see Table 1).

The chelator-containing liposomes consisted of DSPC/CHOL/DSPE-PEG₂₀₀₀ inthe molar ratio 50:40:10. An isotonic HEPES buffer (10 mM HEPES, 150 mMNaCl, pH 7.4, 300 mOsm/L) was used as exterior buffer, and a HEPESbuffer containing chelator (10 mM DTPA or DOTA, 10 mM HEPES, 150 mMNaCl, pH 7.4) was used as interior buffer. Approximately 10 μL ofradioactive ¹⁷⁷LuCl₃, InCl₃ or ^(99m)Tc pertechnetat was added topurified chelator-containing liposomes (500 μL) followed by incubationfor 60 min at 50-55° C. The radioactive ¹⁷⁷LuCl₃ and ¹¹¹InCl₃ waspurchased from Pelkin Elmer, Denmark, and the ^(99m)Tc pertechnetat waskindly provided from Køge Hospital, radiology department, Denmark.

TABLE 1 Loading efficiencies of ⁶⁴Cu²⁺, ¹¹¹In³⁺, ¹⁷⁷Lu³⁺ and ^(99m)TcO₄⁻ into liposomes consisting of DSPC/CHOL/DSPE-PEG₂₀₀₀ (50:40:10) with 10mM chelator entrapped. The loading was carried out for 60 min at 50-55°C. without using ionophore and evaluated by SEC. Loading efficiencyRadionuclide Entrapped chelator [%] ⁶⁴Cu²⁺ DOTA 98 ⁶⁴Cu²⁺ DTPA 95¹¹¹In³⁺ DOTA 96 ¹⁷⁷Lu³⁺ DOTA 81/88* ^(99m)TcO₄ ⁻ DTPA  0 *loading for 4hours at 65° C.

The results in Table 1 indicate that the loading method leads to cationpermeability (⁶⁴Cu²⁺, ¹⁷⁷Lu³⁺ and ¹¹¹In³⁺) of liposome compositions withhighly favourable loading kinetics.

To characterize and optimize the loading methods of the presentinvention different experiments were performed and the followingparameters were tested; (1) Effect of lipid composition, (2) Effect oflipid concentration and entrapped volume, (3) Effect of free fattyacids, (4) Effect of monovalent ions (Na⁺, Cl⁺) and competing divalention (Ca²⁺), (5) Effect of chelating components on the exterior, (6)Effect of interior liposomal pH, (7) Phase behavior and effect ofloading temperature, (8) Loading kinetics and influence of temperature,(9) Hyper- and hypo-osmotic pressure and (10) Cu²⁺ loading kinetics withand without ionophore.

(1) Effects of Lipid Composition

Liposome compositions within this study are formed fromphosphatidylcholines (PC) as 1,2-disteraroyl-sn-glycero-3-phosphocholine(DSPC) and polyethyleneglycol (PEG) derivatized phosphatidylethanolamine as1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000](DSPE-PEG₂₀₀₀). Besides DSPC and DSPE-PEG₂₀₀₀, cholesterol isincorporated in the membrane. Generally, cholesterol increases bilayerthickness and fluidity while decreasing membrane permeability, and doesnot add any charge to the membrane. DSPE-PEG₂₀₀₀ is negatively charged.The liposomes evaluated here were composed of DSPC, cholesterol andDSPE-PEG₂₀₀₀. The overall membrane potential of the liposome composition(evaluated via the zeta-potential) is slightly negative for a liposomewith the lipid composition DSPC/CHOL/DSPE-PEG₂₀₀₀ 50:40:10;approximately −15 mV when measured in 10 mM HEPES buffer supplementedwith 300 mM glucose, pH 7.4, 336 mOsm/L (Table 2). The differentliposome compositions in Table 2 were loaded with ⁶⁴Cu²⁺ by incubatingliposome compositions with ⁶⁴Cu²⁺ for 60 min at 50-55° C. without usingionophore and evaluated by SEC.

TABLE 2 Loading efficiencies of ⁶⁴Cu²⁺ into different liposomecompositions containing 10 mM DOTA encapsulated. Loading was performedwithout using ionophore and by incubating for 60 min at 50-55° C. andevaluated by SEC. Liposome composition Loading efficiency (% molarratio) [%] Zeta potential [mV] DSPC/CHOL/DSPE-PEG₂₀₀₀ 98 −16.2*/−2.8**(50:40:10) DSPC/CHOL/DSPE-PEG₂₀₀₀ 98 −15.2*/−6.3** (55:40:5) DSPC/CHOL(60:40) 92 −11.5*/+2.9** DSPC 92  −8.6*/+12.9** *Zeta potential wasmeasured in a 10 mM HEPES buffer supplemented with 300 mM glucose, pH7.4, 336 mOsm/L. **Zeta potential was measured in a 10 mM HEPES buffersupplemented with 300 mM glucose and 1 mM CaCl₂, pH 7.4, 339 mOsm/L.

The negative membrane potential could influence the high loadingefficiencies of the cationic metal ions into the liposomes (Table 1). Inorder to evaluate this, loading experiments were conducted on neutralliposome compositions excluding DSPE-PEG₂₀₀₀ only consisting of pureDSPC or a mixture of DSPC and cholesterol in the molar ratio 60:40. Allliposome compositions contained high chelator concentrations (DOTA, 10mM) in the interior. The chelator-containing liposomes were added to adried vial with radioactive ⁶⁴CuCl₂ and incubated for 60 min at 50-55°C. High ⁶⁴Cu²⁺ loading efficiencies were observed with all liposomescompositions (see Table 2).

(2) Effect of Lipid Concentration and Entrapped Volume

Classical means of entrapping drugs (known as loading) into liposomesinvolves encapsulating the desired drug during the preparation of theliposomes (passive entrapment). Passive entrapment techniques are lessefficient in encapsulating drugs or other entities compared to activeloading methods (wherein the metal is loaded after preparation of theliposomes). In passive entrapment, the drug or the radionuclide issimply included in the buffer solution from which the liposomes areformed. The liposome size is highly important for passive loading, aspassive entrapment strongly depends on the entrapped aqueous volume ofthe liposomes.

Here, a passive entrapment of ⁶⁴Cu²⁺ was tested. The passive entrapmentwas carried out as described in the following: A lipid-film was made bymixing the lipids (DSPC, cholesterol and DSPE-PEG₂₀₀₀ in the molar ratioof 55:40:5 with a lipid concentration of 50 mM) in chloroform and driedunder a gentle stream of nitrogen. Organic solvent residues were removedunder reduced pressure overnight. The lipid-film was the dispersed byadding an aqueous solution—a HEPES buffer (10 mM, 150 mM NaCl, pH 7.4)containing the chelating-agent, DOTA, adjusted to either pH 4.0 or pH7.4 with a concentration of 10 mM together with radioactive ⁶⁴CuCl₂. Thesolution was passively loaded with ⁶⁴Cu²⁺ by hydrating the solution at65° C. for 60 min. Passively ⁶⁴Cu-loaded multi-lamellar vesicles (MLVs)were sized to large unilamellar vesicles (LUVs)⁶⁴Cu-loaded by multipleextrusions through 100 nm polycarbonate filters using a automateddispenser system applicable for radioactive samples and the loadingefficiency was evaluated by SEC. An encapsulation efficiency of 6.25%was obtained in a 100 nm sized liposome solution composed of DSPC,cholesterol and DSPE-PEG₂₀₀₀ in the molar ratio of 55:40:5 with a lipidconcentration of 50 mM. From this the following conclusion was made;˜0.14% ⁶⁴Cu²⁺ is passively encapsulated or associated with the membraneper mM lipid in 100 nm liposomes. This assumption is consistent withestimates of the entrapped volume and encapsulation degree of 100 nmsized unilamellar liposomes:

$\begin{matrix}\left( {{\left( {V_{entrap}/V_{tot}} \right)/C_{lip}} = {{{Na} \cdot a_{lip} \cdot \frac{R}{6}} \sim {0.20{\%/{mM}}}}} \right. & {{Equation}\mspace{14mu} 5}\end{matrix}$

V_(entrap) and V_(tot) being the entrapped and total volume, C_(lip) isthe lipid concentration, Na is the Avogadro's number, a_(lip)=40 Å² isthe approximate average cross-sectional area of the lipid compositionused and R is the liposome radius.

To test if the method of loading metal cations into preformed liposomesis proportional to the lipid concentration and the entrapped volume, theuptake of radioactive ⁶⁴Cu²⁺ into neutral membrane compositionsconsisting of a mixture of DSPC and CHOL in the molar ratio 60:40without any chelating agent encapsulated was observed. Chelator-freeliposomes were prepared as follows: The lipids (DSPC and CHOL) weremixed in chloroform and dried to a lipid-film under a gentle stream ofnitrogen. Organic solvent residues were removed under reduced pressureovernight. The lipid-film was dispersed by adding an aqueous solution—aHEPES buffer (10 mM, 150 mM NaCl, pH 7.4) and the osmolarity wasmeasured to be 300 mOsm/L. The solution was then hydrated at 65° C. for60 min. Multi-lamellar vesicles (MLVs) were sized to large unilamellarvesicles (LUVs) by multiple extrusions through 100 nm polycarbonatefilters using a mini-extruder. The buffer used in this experiment wasthe same used in all other experiments; an isotonic HEPES buffer (10 mMHEPES, 150 mM NaCl, pH 7.4, 300 mOsm/L). The liposomes were incubatedwith ⁶⁴Cu²⁺ for 60 min at 50-55° C. and evaluated by SEC. The liposomalloaded radioactivity was 0.75% when the lipid concentration was low (5mM) and 5.3% when the lipid concentration was 10-fold higher (50 mM)(see Table 3).

TABLE 3 Percent radioactivity associated to the liposome compositionswithout chelator encapsulated. The incubations were carried out for 60min at 50-55° C. without using ionophore and evaluated by SEC. LoadingLiposome composition Lipid concentration efficiency Radionuclide (%molar ratio) [mM] [%] ⁶⁴Cu²⁺ DSPC/CHOL (60:40) 50 5.3 ± 1.0 ⁶⁴Cu²⁺DSPC/CHOL (60:40) 5 0.75 ¹¹¹In³⁺ DSPC/CHOL/DSPE- 50 4 PEG₂₀₀₀ (50:40:10)

It is clear that the loading efficiency of passive loading usingtemperatures of 50-55° C. is significantly lower (6.25%) than theloading efficiency obtained by using the loading methods of the presentinvention (e.g. Table 1 and 2). The results in Table 3 also indicatethat loading of ⁶⁴Cu²⁺ into the liposomes without chelator encapsulatedusing the method of the present invention is proportional to theentrapped volume and/or the lipid concentration of the liposomes,indicating that loading/association of Cu²⁺ into preformed liposomes canoccur unassisted by an entrapped chelator. The hypothesis was alsotested with the trivalent metal ion, ¹¹¹In³⁺ in showing similar trendsas for ⁶⁴Cu²⁺ (see Table 3). Either the metal ions are trapped ortransported passively in the aqueous phase of preformed liposomes due tosimple transmembrane ion equilibrium or the metal ions are associated tothe lipids in the membrane of the liposome. The metal ions could bind toor associate to the phosphate moiety in the polar head group of PC. Theresults in Table 3 clearly demonstrate a correlation between the lipidconcentration and/or the entrapped liposomal volume and the Cu²⁺ andIn³⁺ ions association to or transport into the liposomes.

(3) Effect of Free Fatty Acids

Free fatty acids (FFA) are known to diffuse (or flip-flop) rapidlyacross phospholipid bilayers in their protonated form. However, whetherflip-flop through the hydrophobic core of the bilayer or desorption fromthe membrane into the aqueous phase is the rate-limiting step in FFAtransport through membranes is still debated. Nevertheless, FFAs arewell known to have a destabilizing effect on some liposomal membranesenhancing the permeability of membranes and facilitating the passage ofentities over the membrane; however, exceptions are known where FFAsstabilize the gel state of fully saturated lipid membranes. The additionof FFA to lipid bilayer solutions such as liposomes have been shown todramatically enhance membrane permeability in the presence of e.g.palmitic acid and Ca²⁺ ions [Agafonov et al., BBA, 1609:153-160, 2003].To evaluate if the high radionuclide loading into the aqueous phase ofliposomes without the use of ionophores found for the present invention,could be dependent on the presence of FFA that enhance the trans-bilayerdiffusion rate of free metal ions (acyl phospholipids contain smallimpurities of FFA), the ⁶⁴Cu²⁺ loading efficiency was measured fornon-FFA containing liposomal membranes.1,2-Di-O-Hexadecyl-sn-Glycero-3-phosphocholine (1,2-Di-O-DPPC) was usedas FFA free lipid component replacing DSPC in the liposome composition(see FIG. 5).

The chelator-containing non-FFA containing liposomes were prepared asdescribed in Example I: Preparation of liposome composition containingchelating-agent, using 1,2-Di-O-Hexadecyl-sn-Glycero-3-phosphocholine(1,2-Di-O-DPPC) and CHOL as vesicle-forming components in the molarratio 60:40. The chelator-free non-FFA containing liposomes wereprepared as described in the above section (2) using1,2-Di-O-Hexadecyl-sn-Glycero-3-phosphocholine (1,2-Di-O-DPPC) and CHOLas vesicle-forming components in the molar ratio 60:40.

The chelator-containing (10 mM DOTA) non-FFA containing liposomes orchelator-free non-FFA containing liposomes were added to a dried vialwith radioactive ⁶⁴CuCl₂ and incubated for 60 min at 50-55° C. andevaluated by SEC. A high loading of ⁶⁴Cu²⁺ into the interior of thechelator-containing non-FFA containing liposomes was observed (Table 4)with chelator-free non-FFA containing liposomes serve as a control.

TABLE 4 Loading efficiencies of ⁶⁴Cu²⁺ into liposome compositionscontaining 1,2-Di-O-Hexadecyl-sn-Glycero-3-phosphocholine(1,2-Di-O-DPPC) and CHOL in the molar ratio 60:40 with and without 10 mMDOTA encapsulated. The loading were carried out for 60 min at 50-55° C.without using ionophore and evaluated by SEC. Lipid Loading Liposomecomposition concentration efficiency Chelator (% molar ratio) [mM] [%]With 1,2-Di-O-DPPC/CHOL (60:40) 10 93 Without 1,2-Di-O-DPPC/CHOL (60:40)50 6 1,2-Di-O-DPPC: 1,2-Di-O-Hexadecyl-sn-Glycero-3-phosphocholine

This excludes the possibility that FFAs are inducing the permeability ofthe free metal ions into the liposomes. Non-FFA containing liposomeswithout chelator encapsulated served as a control, and gave similarresults as for DSPC/CHOL (60:40) without encapsulated chelators (Table3). The conclusion is that liposomes both with and without FFA in themembrane can be used in the present invention.

(4) Effect of Monovalent Ions (Na⁺, Cl⁻) and Competing Divalent Ions(Ca²⁺)

In a study by Hauser and Dawson it was observed that monovalent ionslike Na⁺ and K⁺ were only effective at displacing Ca²⁺ when they werepresent at a concentration about 10⁴ times that of Ca²⁺[Hauser andDawson, J. Biochem., 1:61-69, 1967], which agrees with the predictionsof the double layer theory [Lyklema, ISBN:0-12-460530-3, 5:3.208, 1995].The double layer is a structure that appears on the surface of an objectwhen it is placed into liquid containing ions. The object might be asolid particle such as a nanoparticle or liposome. In the first layer,the surface charge (either positive or negative) comprises ions adsorbeddirectly onto the object. The second layer is composed of ions attractedto the surface charge via Coulomb force, thereby electrically screeningthe first layer. This second layer is loosely associated with thenanoparticle, because it is made of free ions which move in the liquidunder the influence of electric attraction and thermal motion ratherthan being firmly anchored.

As reported above, 5.3% radioactivity was associated/loaded to theliposomes when an isotonic HEPES buffer (10 mM HEPES, 150 mM NaCl, pH7.4, 300 mOsm/L) was used (Table 3), but if no monovalent ions (Na⁺ andCl⁻) were added (10 mM HEPES, pH 7.4, 5 mOsm/L), 11% radioactivity wasassociated with the DSPC/CHOL membrane (50 mM) (see Table 5).

TABLE 5 Loading efficiencies of ⁶⁴Cu²⁺ into liposome compositionswithout chelator encapsulated. The liposome composition consisted oflipid components DSPC and CHOL in the molar ratio 60:40 with a totallipid concentration of 50 mM. The incubations were carried out for 60min at 50-55° C. without using ionophore and loading was subsequentlyevaluated by SEC. Loading External and internal buffer solutionefficiency [%] 10 mM HEPES (pH 7.4, 5 mOsm/L) 11 10 mM HEPES, 150 mMNaCl (pH 7.4, 300 mOsm/L) 5.3 ± 1.0 10 mM HEPES, 150 mM NaCl, 10 mMCaCl₂ 3 ± 1 (pH 7.4, 315 mOsm/L)

This is in agreement with double layer theory predicting a strongerinteraction between the negatively charged lipid membrane and Cu²⁺ asthe screening is reduced by the removal of NaCl. In order tosubstantiate this point, we repeated the loading experiment with theDSPC/CHOL membrane (50 mM) at higher ionic strength adding 10 mM of Ca²⁺(using 10 mM HEPES, 150 mM NaCl, 10 mM CaCl₂, pH 7.4, 315 mOsm/L). Asignificant reduction (3%) of radioactivity was associated to themembrane (see Table 5), indicating that monovalent ions such as Na⁺ anddivalent ions as Ca²⁺ effectively displace ⁶⁴Cu²⁺ at the membranesurface thereby reducing the ⁶⁴Cu²⁺ loading rate. This observation isfurthermore in agreement with previous investigations on interactions ofdivalent cations such as Ca²⁺ and Zn²⁺ with phospholipid membranes[Altenbach and Seelig, Biochemistry, 23:3913-3920, 1984; Binder et al.,Bio-phys. Chem., 90:57-74, 2001; Huster et al., Biophys. J.,78:3011-3018, 2000]. In addition the study by Binder and Zschörnig[Binder and Zschörnig, Chem. Phys. Lipids, 115:39-61, 2002] showed thatCa²⁺ clearly binds to the lipid headgroup of pure POPC lipid bilayers.From the results reported here it is suggested that the primary bindingof the metal cation, Cu²⁺ to the membrane, is reduced by chargescreening effects by mono- and divalent ions such as Ca²⁺ and Na⁺.

Importantly, it can be seen from the results in Table 5 that the loadingmethods of the present invention of Cu²⁺ (divalent ions, radioactive andnon-radioactive, as well as radioactive trivalent cations, ¹⁷⁷Lu³⁺ and¹¹¹In³⁺) into chelator-containing liposomes can be conducted both inpresence or absence of Ca²⁺, Na⁺ and cr.

(5) Effect of Chelating Components

The distribution between, and binding of free metal ions (radionuclides)to, various components on the outside of the liposomes (such asun-removed chelator, buffer molecules etc.) are important in determiningthe chemical activity of the free metal ions with respect totrans-membrane diffusion and overall remote loading kinetics. Whenresidual chelators or other metal binding components are present on theoutside of the liposomes, the loading kinetics and efficiency is lowereddramatically. This was observed when a chelator-containing liposomesolution was spiked with 10⁻⁶ M DOTA prior to incubation. The loadingefficiency was lowered to 2% compared to when no chelator componentswere present on the outside (>95%). To achieve high loading efficiency(for all cations tested (⁶⁴Cu²⁺, ¹⁷⁷Lu³⁺ and ¹¹¹In³⁺)) it is importantto remove residual chelators (e.g. DOTA) from the outside of thechelator-containing liposomes during preparation. The presence ofchelating components on the liposome exterior lowers the cationconcentration (e.g. ⁶⁴Cu²⁺, ¹⁷⁷Lu³⁺ and ¹¹¹In³⁺) in the aqueous phaseand thereby the concentration of the membrane associated fraction, whichleads to a very low loading efficiency within an appropriate time scale(hours).

Besides chelators, buffer components are able to complex metal ions. Itis known that the buffer HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) interacts with Cu²⁺and forms complexes that undergoes alkaline hydrolysis above pH 6,resulting in Cu(OH)₂ precipitation [Sokolowska and Bal, J. Inorg.Biochem., 99:1653-1660, 2005]. It has been proposed that HEPES containsmall impurities that have relatively high affinity for Cu²⁺[Mash andChin, Anal. Chem., 75:671-677, 2003]. Hypothetically, HEPES could act ascarrier molecule of metal ions within the loading methods of the presentinvention, shedding and transporting the ions over the membrane wherethey preferentially bind to the pre-encapsulated chelator.

Since similar high loading efficiencies (>95%) of ⁶⁴Cu²⁺ intochelator-containing liposomes (DSPC/CHOL/DSPE-PEG₂₀₀₀ in the molar ratio50:40:10), were obtained when using other buffers such as phosphatebuffered saline (PBS) and the “non-coordinating” buffer2-(N-morpholino)ethanesulfonic acid (MES), HEPES may not act as carriermolecule of Cu(II) (see Table 6). The preparation of the liposomes wascarried out as described in Example I: Preparation ofchelator-containing liposomes, where HEPES buffer was replace by PBS orMES buffer. The loading was performed for 60 min at 50-55° C. and theloading efficiency was evaluated by SEC.

TABLE 6 Loading efficiencies of ⁶⁴Cu²⁺ into liposome compositions(DSPC/CHOL/DSPE-PEG₂₀₀₀ in the molar ratio 50:40:10) containing 10 mMDOTA encapsulated with different external buffer solutions. Loading wasperformed for 60 min at 50-55° C. without using ionophores and evaluatedby SEC. Loading External buffer solution efficiency [%] 10 mM HEPES, 150mM NaCl (pH 7.4, 300 mOsm/L) 98 ± 2 10 mM MES, 150 mM NaCl (pH 5.9, 300mOsm/L) 95 9.5 mM PBS, 150 mM NaCl (pH 7.4, 300 mOsml/L) 95 ± 3

The present results show that a high loading efficiency is obtainedusing the methods of the present invention with an incubation solutioncomprising different loading buffers,

However, the solubility of dried ⁶⁴CuCl₂ was found to be higher and morerapid in HEPES buffer compared to PBS and sterile water at pH 7.4 at 22°C. temperature, which is convenient for the preparation procedure. Athigher temperatures the solubility of dried ⁶⁴CuCl₂ in HEPES, PBS andsterile water was equal.

(6) Effect of Interior Liposomal pH

An important discovery in liposome loading techniques was thattrans-membrane ion gradients can be generated and utilized to activelyload and encapsulate ionizable drugs in the aqueous liposome lumen [U.S.Pat. Nos. 5,736,155; 5,077,056; and 5,762,957]. The method involvesestablishing a pH gradient across a liposome bilayer such that anionizable drug, to be encapsulated within a liposome, is uncharged inthe external buffer and charged within the aqueous interior. This allowsthe drug to cross the bilayer membrane of the liposome in a neutral formand to be trapped within the aqueous interior of the liposome due toconversion to the charged form. Leakage of drug from actively loadedliposomes has also been found to follow the loss of the proton gradient.

In a previous study on ⁶⁴Cu²⁺ loading into liposomes using the ionophore2-hydroxyquinoline (2HQ) [Petersen et al., Biomaterials, 32:2334-2341,2011], >95% and 70% loading efficiency was observed forchelator-containing liposomes with interior pH of 4.0 and 5.9respectively. The lower degree of loading obtained at pH 5.9 wasexplained by the less favorable exchange of ⁶⁴Cu²⁺ from 2HQ to DOTA.Another ionophore, oxine, was also evaluated, but provided unstableliposomes with respect to ⁶⁴Cu²⁺ retention. This instability (release of⁶⁴Cu²⁺) was explained by the ionophore's ability to dissipate thetransmembrane pH gradient, causing the liposomal interior pH toincrease, which in the case of oxine, resulted in a reduction of theligand exchange by several orders of magnitude. Thus ionophores canfacilitate the release of entrapped metal ions from liposomecompositions.

The influence of interior liposomal pH on the loading efficiency andretention of metal ions was tested with the loading methods of thepresent invention. Chelating-agent (C_(DOTA)=10 mM) was trapped withinthe liposomes (DSPC/CHOL/DSPE-PEG₂₀₀₀ in the molar ratio 50:40:10)adjusted to either pH 4.0 or pH 7.4 as previously described in ExampleI: Preparation of chelator-containing liposomes. The external buffer wasan isotonic HEPES buffer (10 mM HEPES, 150 mM NaCl, pH 7.4, 300 mOsm/L).The incubations of liposomes with ⁶⁴Cu²⁺ were carried out for 60 min at50-55° C. and followingly evaluated by SEC. High loading efficiencies(>95%) of ⁶⁴Cu²⁺ were obtained for both interior liposomal pH (4.0 and7.4) (see Table 7).

TABLE 7 Loading efficiencies of ⁶⁴Cu²⁺ into liposome compositions(DSPC/CHOL/DSPE-PEG₂₀₀₀ in the molar ratio 50:40:10) containing 10 mMDOTA encapsulated with different interior pH (pH 4.0 or 7.4). Loadingwas performed for 60 min at 50-55° C. without using ionophores andfollowingly evaluated by SEC. External buffer was an isotonic HEPESbuffer (10 mM HEPES, 150 mM NaCl, pH 7.4, 300 mOsm/L). Internal bufferLoading efficiency pH [%] Leakage [%] 4.0 98 ± 2 <1% 7.4 98 ± 2 <1%

In addition, ⁶⁴Cu²⁺ loaded liposomes were tested for radionuclideretention by incubating the liposome solutions for 24 h at 37° C. and20° C. Additionally, stability in human serum of the liposome solutionswas tested by mixing human serum and liposome solution (1:1) at 37° C.for 24 h. No release of entrapped radionuclide, ⁶⁴Cu²⁺, was observedfrom any of the liposome solutions (see Table 7).

From the results obtained provided in Table 7 it is clear that theinterior pH can easily be raised to pH 7.4 without any influence onloading efficiency or radionuclide retention. Thus the loading method ofthe present invention is not dependent on any pH gradient across themembrane. An interior pH 7.4 of the liposome is preferable due topossible lipid hydrolysis at lower pH such as pH 4.0. The shelf-life ofthe chelator-containing liposomes is therefore also significantlyprolonged when using interior pH 7.4 compared to pH 4.0.

(7) Phase Behavior and Effect of Loading Temperature

Conventional approaches to liposome formulation dictate inclusion ofsubstantial amounts (e.g. 30-45 mol %) of cholesterol or equivalentmembrane rigidifying agents (such as other sterols). Generally,cholesterol increases the bilayer thickness and fluidity whiledecreasing membrane permeability of the liposome. For example, it hasbeen reported that including increasing amounts of cholesterol inphosphatidylcholine (PC) containing liposomes decreased the leakage ofcalcein (a fluorescent marker compound) from liposomes in the presenceand absence of an osmotic gradient [Allen, et al. Biochim, Biophys.Acta, 597:418-426, 1980]. Another feature of adding cholesterol to lipidbilayers is the formation of a liquid-ordered phase inheriting thestability properties of the liquid-crystaline phase and mobility of thefluid phase. When the DSPC bilayer is supplemented with more than ˜35mol % of cholesterol, the main phase transition is completely abolished,and the membrane can be considered to exist in a liquid-ordered phaseover a wide temperature range. From differential scanning calorimetry(DSC) experiments it is observed that the liposomal membrane composed ofDSPC, CHOL and DSPE-PEG₂₀₀₀ in the molar ratio of 50:40:10 does notexhibit any thermal transitions in the range 45-60° C. and thus existsin a single (liquid-ordered like) phase within this temperature range(FIG. 6). Still as shown in FIG. 3, the loading efficiency of ⁶⁴Cu²⁺into liposome compositions without the use of an ionophore wasincreasing with increasing temperature reaching a high loadingefficiency (96.7%±1.0%) at 50-55° C. for 60 min. The efficienciespresented here for loading without use of ionophores are proportionalwith the increasing temperatures, however, since no phase transitiontemperature occurs in the liposome composition (FIG. 6), the augmentedloading efficiencies are not caused by a phase transition behavior.

(8) Loading Kinetics and Loading Temperature

The kinetics of ⁶⁴Cu²⁺ loading into chelator-containing liposomes(DSPC/CHOL/DSPE-PEG₂₀₀₀ in the molar ratio 50:40:10), were examined byradio thin layer chromatography (radio-TLC) as the ratio betweencomplexed ⁶⁴Cu (e.g. ⁶⁴Cu-DOTA) and the total ⁶⁴Cu amount (sum ofcomplexed (⁶⁴Cu-DOTA) and free ⁶⁴Cu²⁺) as function of time. The loadingexperiments were carried out in a reaction vial at 30° C., 40° C. and50° C. and 2 μL samples were spotted on TLC plates at different timepoints. Thus when ⁶⁴Cu²⁺ is loaded into the liposomes, the metal ionbinds preferentially to DOTA and ⁶⁴Cu-DOTA complex is formed. The TLCplates were run in an organic eluent (10% ammonium acetate:methanol(50:50)) where ⁶⁴Cu-DOTA complexes were separated from free ⁶⁴CuCl₂. Theretention factor (R_(f)) of ⁶⁴Cu-DOTA was approximately 0.3 while ionic⁶⁴Cu²⁺ remained on the origin (R_(f)=0). When the liposome samples werespotted on TLC plates, the liposomes immediately dry out and theinterior (⁶⁴Cu-DOTA) runs on the TLC plate. The ratio between theinterior ⁶⁴Cu-DOTA complex and the total ⁶⁴Cu amount (sum of complexed(⁶⁴Cu-DOTA) and free ⁶⁴Cu²⁺) was calculated as the loading efficiency(defined in equation 1). As a control, free ⁶⁴Cu²⁺ was spotted on a TLCplate followed by non-radioactive chelator-containing liposomes on topof ⁶⁴Cu²⁺. This control was done to eliminate an erroneous estimation of⁶⁴Cu²⁺ and DOTA complexation occurring on the TLC plate. Since no⁶⁴Cu-DOTA peak was present on the TLC plate, no complexation isoccurring on the TLC plate.

The loading of metal ions into liposomes can be divided into severalsteps including: (i) binding/coordination/adsorption of the ion to thelipid membrane, (ii) trans-bilayer ion diffusion and (iii) binding ofions to the chelator. In the current loading procedure the lipid andchelator are in large excess compared to the ⁶⁴Cu²⁺ and the kineticsthus only depends on the ⁶⁴Cu²⁺ concentration. The rate ofcoordination/binding of Cu²⁺ to the membrane is rapid (likely to bediffusion limited) and binding of Cu²⁺ to DOTA occurs on timescale ofseconds (verified by radio-TLC) rendering trans-membrane ion diffusionas the most probable rate limiting step. In general, the rate oftrans-membrane diffusion will depend on the concentration gradient ofthe transported entity (according to Ficks 1^(st) law), the membranephase state (gel, fluid or liquid-ordered) and physicochemical(hydrophilicity vs. hydrophobicity) properties of the transportedentity. These arguments substantiate the first order equation (Equation6) presented below. The loading kinetics (example shown in FIG. 7-8) canbe characterized by the equation

$\begin{matrix}{{\% \mspace{14mu} {load}} = {\frac{A_{{Cu} - {chelator}}}{A_{Cu} + A_{{Cu} - {chelator}} + A_{{Cu}{({ionophore})}}} = {a\left( {1 - {b\; ^{- {ct}}}} \right)}}} & {{Equation}\mspace{14mu} 6}\end{matrix}$

where A_(Cu), A_(Cu-chelator) and A_(Cu(ionophore)) denote the TLCactivity of the ⁶⁴Cu²⁺, ⁶⁴Cu-DOTA and ⁶⁴Cu(2HQ)₂ specie. The fittingparameter a, describes the plateau level (a˜100% if loading proceedsaccording to 1^(st) order kinetics), b describes offset and uncertaintyin t (b=1 when offset and uncertainties in t are small) and c describesthe loading rate. By fitting of equation 7, each loading profile can becharacterized by: (i) the initial velocity:

v _(ini) =a·b·c  (equation 7),

(ii) the time required to reach 95% loading:

t _((95%))=−ln((1−(95%)/a)/b)/c  (equation 8),

and (iii) the degree of loading reached at 60 min (% load_(1h)). Thelatter is directly comparable to the loading degree achieved using themethod based on SEC (presented in FIG. 3 and Tables 1, 2, 6 and 7).

The first order rate constant (c) depends on different parameters suchas temperature (see FIG. 7-8) and osmolarity (see next section) at whichthe loading is conducted. The initial velocity (v_(ini)), t_((95%)) and% load_(1h) is given in Table 8 for a set of loading conditions.

TABLE 8 Kinetic parameters for loading conducted at 30° C., 40° C. and50° C., without ionophore for iso-osmotic and hyper-osmotic conditions,and with ionophore (2HQ). The kinetics are characterized by the initialvelocity, v_(ini), the time required to achieve 95% loading, t_((95%))and the loading efficiency obtained after 60 min, % load_(1 h). Allparameters were derived from radio-TLC measurements shown in FIG. 7-8.t_((95%)) v_(ini) [%/min] [min] % load_(1 h) [%] Iso-osmotic 30° C. 0.6220 33 40° C. 3 * 62 50° C. 23 18 99 Hyper-osmotic 30° C. 0.9 240 42 40°C. 7 * 86 50° C. 51 9 99 With ionophore (2HQ) 30° C. 3 80 82 40° C. 7 6094 50° C. 100 6 100 * Extrapolation not possible

The loading efficiency of ⁶⁴Cu²⁺ into liposomes at 50° C. at iso-osmoticconditions (FIG. 8) displays a rapid initial rate which graduallydeclines and saturates as function of time. Upon lowering of thetemperature the initial velocity is decreased significantly (Table 8)and the time required for loading 95% is increased from 30 min toseveral hours (at iso-osmotic loading). Similar temperature effects areobserved for loading at hyper-osmotic conditions (discussed in section9) and for ionophore assisted loading (discussed in section 10).

(9) Hyper- and Hypo-Osmotic Pressure

In order to investigate whether hyper-osmotic conditions increase theloading rate and loading efficiency of metal ions, loading experimentsof ⁶⁴Cu²⁺ into chelator-containing liposomes having a hyper-osmotic(Δ(mOsm/L)=+75), a hypo-osmotic (Δ(mOsm/L)=−40) as well as aniso-osmotic gradient (Δ(mOsm/L)=0) (see Table 9) were conducted. Thepreparation of liposomes and loading experiments were performed asdescribed in Example I, except for changes in the osmolarity of thebuffers (see Table 9 below).

TABLE 9 Loading efficiencies of ⁶⁴Cu²⁺ into liposomes consisting ofDSPC/CHOL/DSPE-PEG₂₀₀₀ (50:40:10) using different intra- andextra-liposomal osmolarties. Loading was performed for 60 min at 50-55°C. without using ionophore and evaluated by SEC followingly. *Δ(mOsm/L)(Interior buffer #/Exterior Loading efficiency buffer#) [%]  0 (#1/#3)96 +80 (#2/#3) 98 −40 (#2/#4) 96  0 (#2/#5) 95 *Δ(mOsm/L): differencebetween the internal and external osmolarity liposomal buffersolution. + is higher internal osmolarity and − is lower internalosmolarity. #1: 10 mM DOTA, 10 mM HEPES, 140 mM NaCl, pH 7.4, 295 mOsm/L#2: 10 mM DOTA, 10 mM HEPES, 150 mM NaCl, pH 7.4, 375 mOsm/L #3: 10 mMHEPES, 150 mM NaCl, pH 7.4, 295 mOsm/L #4: 10 mM HEPES, 200 mM NaCl, pH7.4, 415 mOsm/L #5: 10 mM HEPES, 150 mM NaCl, pH 7.4, 75 mM Sucrose, 375mOsm/L

The liposome compositions consisted of DSPC/CHOL/DSPE-PEG₂₀₀₀ in themolar ratio 50:40:10 contained high chelator concentrations (DOTA, 10mM) in the interior. The osmolarity was controlled by adjusting the NaClconcentration or by adding sucrose (see Table 9). The loading efficiency(evaluated after 60 min) conducted at 50-55° C. (results are compiled inTable 9) showed that high loading efficiency of Cu²⁺ (>94%) is obtainedin all cases within the timeframe of the experiment. However, resultsshown in Table 8 indicate a difference in loading rate between thedifferent osmolarities.

⁶⁴Cu²⁺ loading kinetics were in addition evaluated as function of timeat three different temperatures (30° C., 40° C. and 50° C.) and at twoosmotic conditions (iso- and hyper-osmotic) using radio-TLC (FIG. 7-8)with chelator-containing liposomes (DSPC/CHOL/DSPE-PEG₂₀₀₀ in the molarratio 50:40:10). These data (FIG. 7-8) confirm an increased loading rate(initial velocity (v_(ini)) in Table 8) and loading efficiency (%load_(1h) in Table 8) with increased temperature for both iso- andhyper-osmotic conditions. The rate and efficiency was further augmentedwhen loading was conducted at hyper-osmotic conditions when compared toiso-osmotic conditions. The largest change in loading rate and loadingefficiency upon increased osmolarity were observed at 30° C. and 40° C.,whereas little change was found at 50° C.

These results evidence that the loading rate and efficiency can bemodulated significantly by tuning parameters as the temperature and theosmolarity. These parameters are important for the effectiveness of theloading method.

(10) Cu²⁺ Loading Kinetics with and without Ionophore

As shown in FIG. 3, the loading efficiency (% load_(1h) evaluated bySEC) of ⁶⁴Cu²⁺ into liposome compositions when using the ionophore 2HQwas weakly increasing as function of temperature with a maximum loadingefficiency (92.4%±1.4%) at 50-55° C. for 60 min. In contrast, theloading efficiency of ⁶⁴Cu²⁺ into liposome compositions without the useof an ionophore showed a larger increase with augmented temperaturereaching a higher loading efficiency (96.7%±1.0%) at 50-55° C. for 60min compared to the method with ionophore. These results indicate anincrease in loading efficiencies at temperatures below 50° C., whenincubating ⁶⁴Cu²⁺, liposomes and ionophore compared to loading withoutionophore. The concentration of ionophore used in the loadingexperiments in FIG. 3 was 100 μM. Ionophores may be toxic to mammals,and therefore the loaded liposomes need to be purified beforeintravenous injection, which would be a disadvantage in liposomeproduction.

The ⁶⁴Cu²⁺ loading kinetics into the aqueous phase of liposomesconsisting of DSPC/CHOL/DSPE-PEG₂₀₀₀ in the molar ratio 50:40:10 withand without the use of ionophore (C_(2HQ)=100 μM) was compared. Toinvestigate the influence of osmotic pressure on the results of thekinetics, the liposomes were prepared having iso-osmotic conditions. Thesolutions were incubated at different temperatures (30° C., 40° C. and50° C.) and evaluated by radio-TLC as function of time (as describedabove). The radio-TLC results (Table 8) substantiated by results fromFIG. 3 show, that the use of an ionophore: (i) increases the loadingrate (initial velocity (v_(ini))) and loading efficiency (% load_(1h))(below 50° C.) and (ii) lowers the time required to load 95%(t_((95%))). The ionophore assisted loading method furthermore reducesthe activation energy for loading, resulting in smaller changes inloading rate and efficiency as a function of temperature variations whencompared to non-assisted loading.

Previous studies have shown low ion permeability of phospholipidbilayers such as liposome compositions, which has lead to highlyunfavorable loading kinetics for charged ion species [Paula et al.,Biophys. J., 74:319-327, 1998; Hauser et al., Nature, 239:342-344, 1972;Ceh et al., J. Phys. Chem. B, 102:3036-3043, 1998; Mills et al.,Biochim. Biophys. Acta, 1716:77-96, 2005; Papahadjopoulos et al.,Biochim. Biophys. Acta, 266:561-583, 1971; Puskin, J. Membrane Biol,35:39-55, 1977]. The results from the experiments utilizing the loadingmethods of the present invention show the opposite, where charged ionsas ⁶⁴Cu²⁺/⁶³Cu²⁺, ¹¹¹In³⁺ and ¹⁷⁷Lu³⁺ are loaded fast and efficientlyinto chelator-containing liposomes. The results show that the use ofionophores or other lipophilic complexes to increase trans-bilayerdiffusion rates only moderately improves or increases the loading ofdivalent and trivalent cations, as previously thought.

SUMMARY

The present examples show that divalent and trivalent ions (such as forexample ⁶⁴Cu²⁺, ¹¹¹In³⁺ in and ¹⁷⁷Lu³⁺) are passively transportedthrough liposomal membranes encapsulated in high concentrations in theinterior of liposome compositions due to complexation topre-encapsulated chelators.

REFERENCES

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1-38. (canceled)
 39. A kit of parts for preparation of a liposome loadedwith a metal cation, said kit comprising: a) a liposome compositioncomprising a vesicle forming component and a non-lipophilic chelatorenclosed by the vesicle forming component; and a metal cation-containingcomposition for loading into the liposome, wherein the kit of parts doesnot comprise an ionophore capable of transporting the metal cationacross a lipid bilayer.
 40. (canceled)
 41. The kit of parts of claim 39,wherein the metal cation-containing composition comprises two or morecopper radionuclides (⁶¹Cu, ⁶⁴Cu, and ⁶⁷Cu). 42-46. (canceled)
 47. Thekit of parts of claim 39, wherein the vesicle forming componentcomprises one or more amphiphatic compounds selected from the groupconsisting of HSPC, DSPC, POPC, DPPC, CHOL, DSPE-PEG-2000 andDSPE-PEG-2000-TATE.
 48. (canceled)
 49. The kit of parts of claim 39,wherein said chelator is selected from the group consisting of1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA),1,4,8,11-15 tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA),1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methanephosphonic acid)(DOTP), cyclam and cyclen.
 50. (canceled)
 51. The kit of parts of claim39, wherein the metal cations are two or more radionuclides selectedfrom the group consisting of ⁶⁴Cu and ⁶⁷Cu, ⁶¹Cu and ⁶⁷Cu, ⁶⁴Cu and ⁹⁰Y,⁶⁴Cu and ¹¹⁹Sb, ⁶⁴Cu and ²²⁵Ac, ⁶⁴Cu and ¹⁸⁸Re, ⁶⁴Cu and ¹⁸⁶Re, ⁶⁴Cu and²¹¹At, ⁶⁴Cu and ⁶⁷Ga, ⁶¹Cu and ¹⁷⁷Lu, ⁶¹Cu and ⁹⁰Y, ⁶¹Cu and ¹¹⁹Sb, ⁶¹Cuand ²²⁵Ac, ⁶¹Cu and ¹⁸⁸Re, ⁶¹Cu and ¹⁸⁶Re, ⁶¹Cu and ²¹¹At, ⁶¹Cu and⁶⁷Ga, ⁶⁷Cu and ¹⁷⁷Lu, ⁶⁷Cu and ⁹⁰Y, ⁶⁷Cu and ¹¹⁹Sb, ⁶⁷Cu and ²²⁵Ac, ⁶⁷Cuand ¹⁸⁸Re, ⁶⁷Cu and ¹⁸⁶Re, ⁶⁷Cu and ²¹¹At, ⁶⁸Ga and ¹⁷⁷Lu, ⁶⁸Ga and ⁹⁰Y,⁶⁸Ga and ¹¹⁹Sb, ⁶⁸Ga and ²²⁵Ac, ⁶⁸Ga and ¹⁸⁸Re, ⁶⁸Ga and ¹⁸⁶Re, ⁶⁸Ga and²¹¹At, and ⁶⁸Ga and ⁶⁷Cu.
 52. The kit of parts of claim 39, wherein theliposome composition further comprises a targeting moiety. 53.(canceled)
 54. The kit of parts of claim 39, wherein the interior pH ofthe liposome is within the range of 4 to 8.5. 55-61. (canceled)
 62. Thekit of parts of claim 39, wherein said cation is a divalent cation or atrivalent cation.
 63. The kit of parts of claim 39, wherein the metalcation-containing composition comprises one or more radionuclidesselected from the group consisting of Copper (⁶¹Cu, ⁶⁴Cu, and ⁶⁷Cu),Indium (¹¹¹In), Technetium (^(99m)Tc), Rhenium (¹⁸⁶Re, ¹⁸⁸Re), Gallium(⁶⁷Ga, ⁶⁸Ga), Strontium (⁸⁹Sr), Samarium (¹⁵³Sm), Ytterbium (¹⁶⁹Yb)Thallium (²⁰¹Tl) Astatine (²¹¹At), Lutetium (¹⁷⁷Lu), Actinium (²²⁵Ac),Yttrium (⁹⁰Y), Antimony (¹¹⁹Sb), Tin (¹¹⁷Sn, ¹¹³Sn), Dysprosium (¹⁵⁹Dy),Cobalt (⁵⁶Co), Iron (⁵⁹Fe), Ruthenium (⁹⁷Ru, ¹⁰³Ru), Palladium (¹⁰³Pd),Cadmium (¹¹⁵Cd), Tellurium (¹¹⁸Te, ¹²³Te), Barium (¹³¹Ba, ¹⁴⁰Ba),Gadolinium (¹⁴⁹Gd, ¹⁵¹Gd), Terbium (¹⁶⁰Tb), Gold (¹⁹⁸Au, ¹⁹⁹Au),Lanthanum (¹⁴⁰La), and Radium (²²³Ra, ²²⁴Ra).
 64. The kit of parts ofclaim 39, wherein the metal cation is a radionuclide selected from thegroup consisting of ⁶¹Cu, ⁶⁴Cu, ⁶⁷Cu, ¹⁷⁷Lu, ⁶⁷Ga, ⁶⁸Ga, ²²⁵Ac, ⁹⁰Y,¹⁸⁶Re, ¹⁸⁸Re, ¹¹⁹Sb, and ¹¹¹In.
 65. The kit of parts of claim 39,wherein the metal cation is a radionuclide selected from the groupconsisting of ⁶¹Cu, ⁶⁴Cu, ⁶⁷Cu, ¹¹¹In, and ¹⁷⁷Lu.
 66. The kit of partsof claim 39, wherein the metal cation is a radionuclide selected fromthe group consisting of ⁶¹Cu, ⁶⁴Cu, and ⁶⁷Cu.
 67. The kit of parts ofclaim 39, wherein the metal cation is selected from the group consistingof Gd, Dy, Ti, Cr, Mn, Fe, Co, and Ni, including divalent or trivalentions thereof.
 68. The kit of parts of claim 39, wherein the metalcation-containing composition comprises a combination of cationsselected from the group consisting of ⁶⁴Cu and Gd(III), ⁶⁴Cu andDy(III), ⁶⁴Cu and Ti(II), ⁶⁴Cu and Cr(III), ⁶⁴Cu and Mn(II), ⁶⁴Cu andFe(II), ⁶⁴Cu and Fe(III), ⁶⁴Cu and Co(II), ⁶⁴Cu and Ni(II), ⁶⁸Ga andGd(III), ⁶⁸Ga and Dy(III), ⁶⁸Ga and Ti(II), ⁶⁸Ga and Cr(III), ⁶⁸Ga andMn(II), ⁶⁸Ga and Fe(II), ⁶⁸Ga and Fe(III), ⁶⁸Ga and Co(II), ⁶⁸Ga andNi(II), ¹¹¹In and Gd(III), ¹¹¹In and Dy(III), ¹¹¹In and Ti(II), ¹¹¹Inand Cr(III), ¹¹¹In and Mn(II), ¹¹¹In and Fe(II), ¹¹¹In and Fe(III),¹¹¹In and Co(II), ¹¹¹In and Ni(II), ^(99m)Tc and Gd(III), ^(99m)Tc andDy(III), ^(99m)Tc and Ti(II), ^(99m)Tc and Cr(III), ^(99m)Tc and Mn(II),^(99m)Tc and Fe(II), ^(99m)Tc and Fe(III), ^(99m)Tc and Co(II), ^(99m)Tcand Ni(II), ¹⁷⁷Lu and Gd(III), ¹⁷⁷Lu and Dy(III), ¹⁷⁷Lu and Ti(II),¹⁷⁷Lu and Cr(III), ¹⁷⁷Lu and Mn(II), ¹⁷⁷Lu and Fe(II), ¹⁷⁷Lu andFe(III), ¹⁷⁷Lu and Co(II), ¹⁷⁷Lu and Ni(II), ⁶⁷Ga and Gd(III), ⁶⁷Ga andDy(III), ⁶⁷Ga and Ti(II), ⁶⁷Ga and Cr(III), ⁶⁷Ga and Mn(II), ⁶⁷Ga andFe(II), ⁶⁷Ga and Fe(III), ⁶⁷Ga and Co(II), ⁶⁷Ga and Ni(II), ²⁰¹Tl andGd(III), ²⁰¹Tl and Dy(III), ²⁰¹Tl and Ti(II), ²⁰¹Tl and Cr(III), ²⁰¹Tland Mn(II), ²⁰¹Tl and Fe(II), ²⁰¹Tl and Fe(III), ²⁰¹Tl and Co(II), ²⁰¹Tland Ni(II), ⁹⁰Y and Gd(III), ⁹⁰Y and Dy(III), ⁹⁰Y and Ti(II), ⁹⁰Y andCr(III), ⁹⁰Y and Mn(II), ⁹⁰Y and Fe(II), ⁹⁰Y and Fe(III), ⁹⁰Y andCo(II), and ⁹⁰Y and Ni(II), wherein said isotope of metal radionuclideappears in any of the existing oxidation states for the metal includingmonovalent cations, divalent cations, trivalent cations, tetravalentcations, pentavalent cations, hexavalent cations and heptavalentcations.
 69. The kit of parts of claim 41, wherein the two or morecopper radionuclides are selected from the group consisting of ⁶¹Cu and⁶⁴Cu; ⁶¹Cu and ⁶⁷Cu; ⁶⁴Cu and ⁶⁷Cu; and ⁶¹Cu, ⁶⁴Cu and ⁶⁷Cu.
 70. The kitof parts of claim 39, wherein said vesicle-forming component comprisesone or more of the compounds selected from the group consisting oflipids, ceramides, sphingolipids, phospholipids, and pegylatedphospholipids.
 71. The kit of parts of claim 39, wherein said chelatoris selected from the group consisting of 1,4,7,10-tetraazacyclododecane([12]aneN4); 1,4,7,10-tetraazacyclotridecane ([13]aneN4);1,4,8,11-tetraazacyclotetradecane ([14]aneN4);1,4,8,12-tetraazacyclopentadecane ([15]aneN4);1,5,9,13-tetraazacyclohexadecane ([16]aneN4);ethylene-diamine-tetraacetic-acid (EDTA); anddiethylene-triamine-penta-acetic acid (DTPA).
 72. The kit of parts ofclaim 39, wherein said chelator is selected from the group consisting of1,4-ethano-1,4,8,11-tetraazacyclotetradecane (et-cyclam);1,4,7,11-tetraazacyclotetradecane (iso-cyclam);1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA);2-(1,4,7,10-tetraazacyclododecan-1-yl)acetate (DO1A);2,2′-(1,4,7,10-tetraazacyclododecane-1,7-diyl)diacetic acid (DO2A);2,2′,2″-(1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid(DO3A); 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methanephosphonicacid) (DOTP); 1,4,7,10-tetraazacyclododecane-1,7-di(methanephosphonicacid) (DO2P); 1,4,7,10-tetraazacyclododecane-1,4,7-tri(methanephosphonicacid) (DO3P); 1,4,8,11-15 tetraazacyclotetradecane-1,4,8,11-tetraaceticacid (TETA); 2-(1,4,8,11-tetraazacyclotetradecane-1-yl)acetic acid(TE1A); 2,2′-(1,4,8,11-tetraazacyclotetradecane-1,8-diyl)diacetic acid(TE2A); ethylene-diamine-tetraacetic-acid (EDTA), anddiethylene-triamine-penta-acetic acid (DTPA).
 73. The kit of parts ofclaim 39, wherein the interior of the liposome has an osmolarity 10-100mOsm/L higher than the osmolarity of the exterior.
 74. The kit of partsof claim 39, wherein the metal cation is a freeze dried salt or anaqueous solution of said cation.